Methods and products for reprogramming cells to a less differentiated state

ABSTRACT

The present invention relates in part to methods for producing tissue-specific cells from patient samples, and to tissue-specific cells produced using these methods. Methods for reprogramming cells using RNA are disclosed. Therapeutics comprising cells produced using these methods are also disclosed.

The present application claims priority to and benefit of U.S.Provisional Application No. 61/566,948, filed on Dec. 5, 2011, U.S.Provisional Application No. 61/569,595, filed on Dec. 12, 2011, and U.S.Provisional Application No. 61/637,570, filed on Apr. 24, 2012, each ofwhich are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates in part to methods for delivering nucleicacids to cells, and to therapeutics comprising cells produced usingthese methods.

BACKGROUND

Nucleic-Acid Transfection

Nucleic acids can be delivered to cells both in vitro and in vivo bypre-complexing the nucleic acids with charged lipids, lipidoids,peptides, polymers or mixtures thereof. Such transfection reagents arecommercially available, and are widely used for delivering nucleic acidsto cells in culture. Cells exposed to transfection reagent-nucleic acidcomplexes may internalize these complexes by endocytosis or other means.Once inside a cell, the nucleic acid can carry out its intendedbiological function. In the case of protein-encoding RNA, for example,the RNA can be translated into protein by the ribosomes of the cell.

Many variables can affect the efficiency of reagent-based transfection,including the structure of the transfection reagent, the concentrationof the nucleic acid, and the complex-formation time. Designing atransfection protocol is made even more difficult by the fact thatadjusting these variables to increase transfection efficiency oftenincreases transfection-associated toxicity. In addition, several commoncomponents of cell-culture media, including serum, some antibiotics, andpolyanions such as dextran sulfate or heparin, can inhibit transfectionand/or cause cell death when cells are transfected in media containingthese components. Thus, the composition of the transfection medium is acritical factor in determining both transfection efficiency andtransfection-associated toxicity.

Serum-Free Cell Culture

Animal sera such as fetal bovine serum (FBS) are commonly used as asupplement in cell-culture media to promote the growth of many types ofcells. However, the undefined nature of serum makes cells that arecontacted with this component undesirable for both research andtherapeutic applications. As a result, serum-free cell-culture mediahave been developed to eliminate the batch-to-batch variability and therisk of contamination with toxic and/or pathogenic substances that areassociated with serum.

The most abundant protein in serum is serum albumin. Serum albumin bindsto a wide variety of molecules both in vitro and in vivo, includinghormones, fatty acids, calcium and metal ions, and small-molecule drugs,and transports these molecules to cells, both in vitro and in vivo.Serum albumin (most often either bovine serum albumin (BSA) or humanserum albumin (HSA)) is a common ingredient in serum-free cell-culturemedia, where it is typically used at a concentration of 1-10 g/L. Serumalbumin is traditionally prepared from blood plasma by ethanolfractionation (the “Cohn” process). The fraction containing serumalbumin (“Cohn Fraction V” or simply “Fraction V”) is isolated, and istypically used without further treatment. Thus, standard preparations ofserum albumin comprise a protein part (the serum albumin polypeptide)and an associated-molecule part (including salts, fatty acids, etc. thatare bound to the serum albumin polypeptide). The composition of theassociated-molecule component of serum albumin is, in general, complexand unknown.

Serum albumin can be treated for use in certain specializedapplications¹⁻³ (US Patent Appl. Pub. No. US 2010/0168000 A1). Thesetreatment processes are most commonly used to remove globulins andcontaminating viruses from solutions of serum albumin, and often includestabilization of the serum albumin polypeptide by addition of theshort-chain fatty acid, octanoic acid, followed byheat-inactivation/precipitation of the contaminants. For highlyspecialized stem-cell-culture applications, using an ion-exchange resinto remove excess salt from solutions of BSA has been shown to increasecell viability³. However, recombinant serum albumin does not benefitfrom such treatment, even in the same sensitive stem-cell-cultureapplications³, demonstrating that the effect of deionization in theseapplications is to remove excess salt from the albumin solution, and notto alter the associated-molecule component of the albumin. In addition,the effect of such treatment on other cell types such as humanfibroblasts, and the effect of such treatment on transfection efficiencyand transfection-associated toxicity have not been previously explored.Furthermore, albumin-associated lipids have been shown to be criticalfor human pluripotent stem-cell culture, and removing these from albuminhas been shown to result in spontaneous differentiation of humanpluripotent stem cells, even when lipids are added separately to thecell-culture medium⁴. Thus, a cell-culture medium containing albuminwith an unmodified associated-molecule component is thought to becritical for the culture of human pluripotent stem cells. Importantly,the relationship between the associated-molecule component of lipidcarriers such as albumin and transfection efficiency andtransfection-associated toxicity has not been previously explored.

Cell Reprogramming

Cells can be reprogrammed by exposing them to specific extracellularcues and/or by ectopic expression of specific proteins, microRNAs,etc.⁵⁻⁹ While several reprogramming methods have been previouslydescribed, most that rely on ectopic expression require the introductionof exogenous DNA, which carries mutation risks. These risks makeDNA-based reprogramming methods undesirable for therapeuticapplications. DNA-free reprogramming methods based on direct delivery ofreprogramming proteins have been reported^(10, 11), however thesetechniques are too inefficient and unreliable for commercial use. Inaddition, RNA-based reprogramming methods have been described¹²⁻¹⁵,however, all previously disclosed RNA-based reprogramming methods areslow, unreliable, and inefficient when applied to adult cells, requiremany transfections (resulting in significant expense and opportunity forerror), can reprogram only a limited number of cell types, can reprogramcells to only a limited number of cell types, require the use ofimmunosuppressants, and require the use of multiple human-derivedcomponents, including blood-derived HSA and human fibroblast feeders.The many drawbacks of previously disclosed RNA-based reprogrammingmethods make them undesirable for both research and therapeutic use.

Cell-Based Therapeutics

Many diseases are caused by the loss of or damage to one or moretissue-specific cells. Methods for treating such diseases by replacingthe lost or damaged cells with cells taken from animals or from one ormore human donors have been described. However, the critical shortage ofdonor cells represents a barrier to the development of cell-basedtherapeutics for most diseases. In addition, therapeutics based on theuse of cells from non-isogenic donors or animals carry a risk ofrejection. As a result, patients receiving such cells must take strongimmunosuppressant drugs, which themselves carry serious side-effects.

SUMMARY OF THE INVENTION

Here we describe reagents and protocols for transfecting andreprogramming cells. Unlike previously reported methods, certainembodiments of the present invention do not involve exposing the cellsto exogenous DNA or to allogeneic or animal-derived materials, makingreagents and cells produced according to the methods of the presentinvention useful for therapeutic applications.

We disclose methods for treating albumin for use in transfection, and weprovide a cell-culture medium for high-efficiency transfection andreprogramming of cells. We further disclose therapeutics comprisingcells that are reprogrammed according to the methods of the presentinvention, including for the treatment of type 1 diabetes, heartdisease, including ischemic and dilated cardiomyopathy, maculardegeneration, Parkinson's disease, cystic fibrosis, sickle-cell anemia,thalassemia, Fanconi anemia, severe combined immunodeficiency,hereditary sensory neuropathy, xeroderma pigmentosum, Huntington'sdisease, muscular dystrophy, amyotrophic lateral sclerosis, Alzheimer'sdisease, and HIV/AIDS.

DETAILED DESCRIPTION OF THE FIGURES

The present invention is illustrated by way of example, and not by wayof limitation, in the figures of the accompanying drawings and in which:

FIG. 1 depicts RNA encoding the indicated proteins, resolved on adenaturing formaldehyde-agarose gel.

FIG. 2 depicts primary human fibroblasts cultured in media containingthe indicated HSA. The cells were transfected once with a mixture of RNAencoding Oct4, Sox2, Klf4, c-Myc-2 (T58A), and Lin28 two days before thepictures were taken.

FIG. 3 depicts primary human fibroblasts cultured in media containingthe indicated HSA. “+RNA” indicates that the cells were transfecteddaily with a mixture of RNA encoding Oct4, Sox2, Klf4, c-Myc-2 (T58A),and Lin28 beginning on day 0. Pictures were taken on day 3. Arrowsindicate areas of morphological changes indicative of reprogramming.

FIG. 4 depicts primary human fibroblasts cultured in media containingthe indicated HSA, and transfected daily with a mixture of RNA encodingthe proteins Oct4, Sox2, Klf4, c-Myc-2 (T58A), and Lin28 beginning onday 0. Pictures were taken on day 2.

FIG. 5 depicts primary human fibroblasts transfected daily with RNAencoding the proteins Oct4, Sox2, Klf4, c-Myc-2 (T58A), and Lin28,beginning on day 0. Pictures were taken on day 10. Large colonies ofcells with a reprogrammed morphology are visible in each picture. Thebottom panel depicts a representative field, showing a high density ofreprogrammed cells, indicating high-efficiency reprogramming.

FIG. 6 depicts primary human fibroblasts transfected and cultured as inFIG. 5, but without using feeders or immunosuppressants. A total of 5transfections were performed. Pictures were taken on day 7. A smallcolony of cells with a reprogrammed morphology is visible in the centerof the picture.

FIG. 7 depicts a reprogrammed cell line one day after colonies werepicked and plated on a basement membrane extract-coated plate.

FIG. 8 depicts a reprogrammed cell line stained for the pluripotentstem-cell markers Oct4 and SSEA4. The panel labeled “Hoechst” shows thenuclei, and the panel labeled “Merge” shows the merged signals from thethree channels.

FIG. 9 depicts primary human fibroblasts transfected and cultured as inFIG. 6. A total of 5 transfections were performed. Pictures were takenon day 7. Several colonies of cells with a reprogrammed morphology arevisible.

FIG. 10A depicts a 1.5 mm-diameter dermal punch biopsy tissue sample.

FIG. 10B depicts a tissue sample harvested as in FIG. 10A, and suspendedat the air-liquid interface of a solution containing an enzyme.

FIG. 10C depicts primary human fibroblasts harvested as in FIG. 10A,dissociated as in FIG. 10B, and plated in a well of a 96-well plate.

FIG. 11 depicts primary human fibroblasts prepared as in FIG. 10C, andreprogrammed using RNA.

FIG. 12 depicts cardiac cells generated by reprogramming primary humanfibroblasts using the methods of the present invention.

DEFINITIONS

By “molecule” is meant a molecular entity (molecule, ion, complex,etc.).

By “RNA molecule” is meant a molecule that comprises RNA.

By “synthetic RNA molecule” is meant an RNA molecule that is producedoutside of a cell, for example, an RNA molecule that is produced in anin vitro-transcription reaction or an RNA molecule that is produced bydirect chemical synthesis.

By “transfection” is meant contacting a cell with a molecule, whereinthe molecule is internalized by the cell.

By “transfection reagent” is meant a substance or mixture of substancesthat associates with a molecule and facilitates the delivery of themolecule to and/or internalization of the molecule by a cell, forexample, a cationic lipid, a charged polymer or a cell-penetratingpeptide.

By “reagent-based transfection” is meant transfection using atransfection reagent.

By “cell-culture medium” is meant a medium that can be used for cellculture, for example, Dulbecco's Modified Eagle's Medium (DMEM) orDMEM+10% fetal bovine serum (FBS).

By “complexation medium” is meant a medium to which a transfectionreagent and a molecule to be transfected are added and in which thetransfection reagent associates with the molecule to be transfected.

By “transfection medium” is meant a medium that can be used fortransfection, for example, Dulbecco's Modified Eagle's Medium (DMEM) orDMEM/F12.

By “recombinant” is meant a protein or peptide that is not produced inanimals or humans, for example, human transferrin that is produced inbacteria, human fibronectin that is produced in an in vitro culture ofmouse cells or human serum albumin that is produced in a rice plant.

By “lipid carrier” is meant a substance that increases the solubility ofa lipid or lipid-soluble molecule in an aqueous solution, for example,human serum albumin or methyl-beta-cyclodextrin.

By “Oct4 protein” is meant a protein that is encoded by the POU5F1 gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, for example, human Oct4 protein (SEQ ID NO:1), mouse Oct4 protein, Oct1 protein, a protein encoded by POU5F1pseudogene 2, a DNA-binding domain of Oct4 protein or an Oct4-GFP fusionprotein. In some embodiments the Oct4 protein comprises an amino acidsequence that has at least 70% identity with SEQ ID NO:1, or in otherembodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ IDNO: 1. In some embodiments, the Oct4 protein comprises an amino acidsequence having from 1 to 20 amino acid insertions, deletions, orsubstitutions (collectively) with respect to SEQ ID NO: 1. Or in otherembodiments, the Oct4 protein comprises an amino acid sequence havingfrom 1 to 15 or from 1 to 10 amino acid insertions, deletions, orsubstitutions (collectively) with respect to SEQ ID NO:1.

By “Sox2 protein” is meant a protein that is encoded by the SOX2 gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, for example, human Sox2 protein (SEQ ID NO:2), mouse Sox2 protein, a DNA-binding domain of Sox2 protein or aSox2-GFP fusion protein. In some embodiments the Sox2 protein comprisesan amino acid sequence that has at least 70% identity with SEQ ID NO:2,or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identitywith SEQ ID NO:2. In some embodiments, the Sox2 protein comprises anamino acid sequence having from 1 to 20 amino acid insertions,deletions, or substitutions (collectively) with respect to SEQ ID NO:2.Or in other embodiments, the Sox2 protein comprises an amino acidsequence having from 1 to 15 or from 1 to 10 amino acid insertions,deletions, or substitutions (collectively) with respect to SEQ ID NO:2.

By “Klf4 protein” is meant a protein that is encoded by the KLF4 gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, for example, human Klf4 protein (SEQ ID NO:3), mouse Klf4 protein, a DNA-binding domain of Klf4 protein or aKlf4-GFP fusion protein. In some embodiments the klf4 protein comprisesan amino acid sequence that has at least 70% identity with SEQ ID NO:3,or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identitywith SEQ ID NO:3. In some embodiments, the klf4 protein comprises anamino acid sequence having from 1 to 20 amino acid insertions,deletions, or substitutions (collectively) with respect to SEQ ID NO:3.Or in other embodiments, the klf4 protein comprises an amino acidsequence having from 1 to 15 or from 1 to 10 amino acid insertions,deletions, or substitutions (collectively) with respect to SEQ ID NO:3.

By “c-Myc protein” is meant a protein that is encoded by the MYC gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, for example, human c-Myc protein (SEQ IDNO: 4), mouse c-Myc protein, 1-Myc protein, c-Myc (T58A) protein, aDNA-binding domain of c-Myc protein or a c-Myc-GFP fusion protein. Insome embodiments the c-Myc protein comprises an amino acid sequence thathas at least 70% identity with SEQ ID NO:4, or in other embodiments, atleast 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO:4. In someembodiments, the c-Myc protein comprises an amino acid having from 1 to20 amino acid insertions, deletions, or substitutions (collectively)with respect to SEQ ID NO:4. Or in other embodiments, the c-Myc proteincomprises an amino acid sequence having from 1 to 15 or from 1 to 10amino acid insertions, deletions, or substitutions (collectively) withrespect to SEQ ID NO:4.

By “reprogramming” is meant causing a change in the phenotype of a cell,for example, causing a β-cell progenitor to differentiate into a matureβ-cell, causing a fibroblast to dedifferentiate into a pluripotent stemcell, causing a keratinocyte to transdifferentiate into a cardiac stemcell or causing the axon of a neuron to grow.

By “reprogramming factor” is meant a molecule that, when a cell iscontacted with the molecule or the cell expresses the molecule, can,either alone or in combination with other molecules, causereprogramming, for example, Oct4 protein.

By “feeder” is meant a cell that is used to condition medium or tootherwise support the growth of other cells in culture.

By “conditioning” is meant contacting one or more feeders with a medium.

By “fatty acid” is meant a molecule that comprises an aliphatic chain ofat least two carbon atoms, for example, linoleic acid, α-linolenic acid,octanoic acid, a leukotriene, a prostaglandin, cholesterol, a resolvin,a protectin, a thromboxane, a lipoxin, a maresin, a sphingolipid,tryptophan, N-acetyl tryptophan or a salt, methyl ester or derivativethereof.

By “short-chain fatty acid” is meant a fatty acid that comprises analiphatic chain of between two and 30 carbon atoms.

By “albumin” is meant a protein that is highly soluble in water, forexample, human serum albumin.

By “associated molecule” is meant a molecule that is non-covalentlybound to another molecule.

By “associated-molecule-component of albumin” is meant one or moremolecules that are bound to an albumin polypeptide, for example, lipids,hormones, cholesterol, calcium ions, etc. that are bound to an albuminpolypeptide.

By “treated serum albumin” is meant serum albumin that is treated toreduce, remove, replace or otherwise inactivate theassociated-molecule-component of the serum albumin, for example, humanserum albumin that is incubated at an elevated temperature, human serumalbumin that is contacted with sodium octanoate or human serum albuminthat is contacted with a porous material.

By “ion-exchange resin” is meant a material that when contacted with asolution containing ions, replaces one or more of the ions with one ormore different ions, for example, a material that replaces one or morecalcium ions with one or more sodium ions.

By “germ cell” is meant a sperm cell or an egg cell.

By “pluripotent stem cell” is meant a cell that can differentiate intocells of all three germ layers (endoderm, mesoderm, and ectoderm) invivo.

By “somatic cell” is meant a cell that is not a pluripotent stem cell ora germ cell, for example, a skin cell.

By “glucose-responsive insulin-producing cell” is meant a cell that,when exposed to a certain concentration of glucose, produces and/orsecretes an amount of insulin that is different from (either less thanor more than) the amount of insulin produced and/or secreted by the cellwhen the cell is exposed to a different concentration of glucose, forexample, a β-cell.

By “hematopoietic cell” is meant a blood cell or a cell that candifferentiate into a blood cell, for example, a hematopoietic stem cellor a white blood cell.

By “cardiac cell” is meant a heart cell or a cell that can differentiateinto a heart cell, for example, a cardiac stem cell or a cardiomyocyte.

By “retinal cell” is meant a cell of the retina or a cell that candifferentiate into a cell of the retina, for example, a retinalpigmented epithelial cell.

By “skin cell” is meant a cell that is normally found in the skin, forexample, a fibroblast, a keratinocyte, a melanocyte, an adipocyte, amesenchymal stem cell, an adipose stem cell or a blood cell.

By “Wnt signaling agonist” is meant a molecule that performs one or moreof the biological functions of one or more members of the Wnt family ofproteins, for example, Wnt1, Wnt2, Wnt3, Wnt3a or2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine.

By “IL-6 signaling agonist” is meant a molecule that performs one ormore of the biological functions of IL-6 protein, for example, IL-6protein or IL-6 receptor (also known as soluble IL-6 receptor, IL-6R,IL-6R alpha, etc.).

By “TGF-β signaling agonist” is meant a molecule that performs one ormore of the biological functions of one or more members of the TGF-βsuperfamily of proteins, for example, TGF-β1, TGF-β3, Activin A, BMP-4or Nodal.

Serum albumin is a common component of serum-free cell-culture media. Ithas now been discovered that serum albumin can inhibit transfection, andthat including untreated serum albumin in a transfection medium atconcentrations normally used in serum-free cell-culture media can resultin low transfection efficiency and/or low cell viability duringtransfection (see Examples). The serum albumin polypeptide binds to awide variety of molecules, including lipids, ions, cholesterol, etc.,both in vitro and in vivo, and as a result, both serum albumin that isisolated from blood and recombinant serum albumin comprise a polypeptidecomponent and an associated-molecule component. It has now beendiscovered that the low transfection efficiency and low cell viabilityduring transfection caused by serum albumin are caused in part by theassociated-molecule component of the serum albumin. It has been furtherdiscovered that transfection efficiency can be dramatically increasedand transfection-associated toxicity can be dramatically reduced bypartially or completely reducing, removing, replacing or otherwiseinactivating the associated-molecule component of serum albumin. Certainembodiments of the invention are therefore directed to a method fortreating a protein to partially or completely reduce, remove, replace orotherwise inactivate the associated-molecule component of the protein.Other embodiments are directed to a protein that is treated to partiallyor completely reduce, remove, replace or otherwise inactivate theassociated-molecule component of the protein.

Certain embodiments are directed to methods for treating a protein bycontacting the protein with one or more molecules that reduce the lowtransfection efficiency and/or low cell viability during transfectioncaused by the protein. Serum albumin has several binding sites that canbind lipids. Contacting serum albumin with the short-chain fatty acid,sodium octanoate (also known as “octanoic acid”, “octanoate”,“caprylate” or “caprylic acid”) was found to reduce the low transfectionefficiency and low cell viability during transfection caused by serumalbumin (see Examples). Other substances that can be used to treat aprotein include: capric acid, lauric acid, myristic acid, palmitic acid,stearic acid, arachidic acid, behenic acid, lignoceric acid, ceroticacid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid,elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid,alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucicacid, docosahexaenoic acid, tryptophan, N-acetyl tryptophan,cholesterol, other fatty acids, and salts, mixtures, fragments, andderivatives thereof. Substances for treating a protein can be puresubstances, well-defined mixtures or complex or undefined mixtures suchas animal-based or plant-based oils, for example, cod-liver oil. Incertain embodiments, a protein is treated after the protein is purified.In other embodiments, a protein is treated before the protein ispurified. In still other embodiments, a protein is treated at the sametime that the protein is purified. In still other embodiments, a proteinis treated, and the protein is not purified.

Incubating a protein at an elevated temperature can cause partial orcomplete denaturation of the polypeptide component of the protein, whichcan reduce or eliminate binding sites that are critical to maintainingthe associated-molecule component of the protein. Certain embodimentsare therefore directed to methods for treating a protein by incubatingthe protein at an elevated temperature. In one embodiment, the proteinis incubated at a temperature of at least 40 C for at least 10 minutes.In another embodiment, the protein is incubated at a temperature of atleast 55 C for at least 30 minutes. In a preferred embodiment, theprotein is contacted with sodium octanoate, and then incubated at 60 Cfor several hours, preferably between 1 hour and 24 hours, morepreferably between 2 hours and 6 hours. In a more preferred embodiment,the concentration of sodium octanoate is between 5 mM and 50 mM, morepreferably between 10 mM and 40 mM. In certain embodiments, the sodiumoctanoate is replaced with or used in combination with at least oneelement of the group comprising: capric acid, lauric acid, myristicacid, palmitic acid, stearic acid, arachidic acid, behenic acid,lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid,sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid,linoelaidic acid, alpha-linolenic acid, arachidonic acid,eicosapentaenoic acid, erucic acid, docosahexaenoic acid, tryptophan,N-acetyl tryptophan, and cholesterol or a salt, mixture, fragment, andderivative thereof.

Glycation and glycosylation are processes by which one or more sugarmolecules are bound to a protein. Glycation and glycosylation can impactthe binding properties of a protein, and serum albumin contains severalpotential glycation sites. Certain embodiments are therefore directed tomethods for treating a protein by glycating or glycosylating theprotein.

Ion-exchange resins, including anion-exchange, cation-exchange, andmixed-bed resins, are routinely used to deionize solutions. Theassociated-molecule component of proteins such as serum albumin oftencomprises ions. Certain embodiments are therefore directed to a methodfor treating a protein by contacting the protein with one or moreion-exchange resins. In a preferred embodiment, the one or moreion-exchange resins includes a mixed-bed resin containing functionalgroups with proton (H+) and hydroxyl (OH−) forms. In another preferredembodiment, the one or more ion-exchange resins includes an indicatorthat changes color as the resin becomes saturated with ions. It isrecognized that, in addition to contacting with one or more ion-exchangeresins, other methods can be used to reduce, remove, replace orotherwise inactivate the associated-molecule component of a protein,including contacting the protein with charcoal, which may be activatedand/or treated with a chemical such as dextran sulfate, dialysis(including dilution resulting in de-association of theassociated-molecule component, whether or not the de-associatedmolecules are subsequently removed from the solution), crystallization,chromatography, electrophoresis, heat treatment, low-temperaturetreatment, high-pH treatment, low-pH treatment, organic-solventprecipitation, and affinity purification.

Certain methods for treating a protein preferentially reduce, remove,replace or otherwise inactivate specific types of molecules. In certainsituations, it is therefore beneficial to combine two or more methodsfor treating a protein to reduce the low transfection efficiency and/orlow cell viability during transfection caused by the protein. Certainembodiments are therefore directed to a method for treating a proteinusing two or more methods to reduce, remove, replace or otherwiseinactivate the associated-molecule component of the protein. In apreferred embodiment, a protein is contacted with one or moreion-exchange resins, and with activated charcoal. In another preferredembodiment, a protein is contacted with sodium octanoate, incubated atan elevated temperature, contacted with one or more ion-exchange resins,and contacted with activated charcoal. In a more preferred embodiment,the protein is serum albumin, and the elevated temperature is at least50 C.

Certain elements of the associated-molecule component of a protein canbe beneficial to cells in culture, and/or to transfection, for example,certain resolvins, protectins, lipoxins, maresins, eicosanoids,prostacyclins, thromboxanes, leukotrienes, cyclopentenoneprostaglandins, and glucocorticoids. Certain embodiments are thereforedirected to a method for treating a protein to reduce, remove, replaceor otherwise inactivate the associated-molecule component of the proteinwithout reducing, removing, replacing or otherwise inactivating one ormore beneficial elements of the associated-molecule component of theprotein. Other embodiments are directed to a method for treating aprotein to reduce, remove, replace or otherwise inactivate theassociated-molecule component of the protein, and further contacting theprotein with one or more molecules comprising one or more beneficialelements of the associated-molecule component of the protein. Stillother embodiments are directed to a method for treating a protein toreduce the low transfection efficiency and/or low cell viability duringtransfection caused by the protein by contacting the protein with one ormore molecules comprising one or more beneficial elements of theassociated-molecule component of the protein. Still other embodimentsare directed to a method for increasing transfection efficiency and/orincreasing cell viability during transfection by contacting a cell withone or more molecules comprising one or more beneficial elements of theassociated-molecule component of a protein. In a preferred embodiment,the protein is contacted with one or more ion-exchange resins orcharcoal, and is further contacted with a glucocorticoid, preferablycortisol, prednisone, prednisolone, methylprednisolone, dexamethasone orbetamethasone. In another preferred embodiment, the cell is contactedwith a glucocorticoid, preferably cortisol, prednisone, prednisolone,methylprednisolone, dexamethasone or betamethasone. In a more preferredembodiment, the cell is transfected, preferably with one or moresynthetic RNA molecules.

Other embodiments are directed to a medium containing a protein that istreated according to the methods of the present invention. In apreferred embodiment, the medium is a transfection medium. In a morepreferred embodiment, the medium also supports efficient transfectionand high cell viability. In certain embodiments, the protein and one ormore molecules that reduce the low transfection efficiency and/or lowcell viability during transfection caused by the protein are addedindependently to the medium. In a preferred embodiment, the protein istreated before being mixed with one or more of the other ingredients ofthe medium. It has now been discovered that, under certain conditions,the concentration of fatty acids/lipids that is commonly used inserum-free cell-culture media is insufficient to treat the large amountof serum albumin that is commonly used in these media. Certainembodiments are therefore directed to a medium that contains a ratio offatty acids/lipids to protein that is sufficient to treat the protein.In a preferred embodiment, the medium contains a standard amount offatty acids/lipids, and a reduced amount of protein, preferably between0.01 g/L and 2 g/L, more preferably between 0.05 g/L and 0.5 g/L.Certain embodiments are directed to a medium containing a protein thatis treated by contacting the protein with one or more ion-exchangeresins. In a preferred embodiment, the protein is treated by contactingthe protein with the one or more ion-exchange resins before the proteinis added to the medium. In a more preferred embodiment, the medium is atransfection medium and the degree of treatment is adjusted to controlthe efficiency of transfection and/or cell viability, preferably toallow transfections at least every 48 hours, more preferably to allowtransfections every 24 hours, more preferably to allow transfectionreagent-nucleic acid complexes to be left in contact with cells forabout 24 hours. Leaving the transfection reagent-nucleic acid complexesin contact with cells for an extended period of time may be desirable inpart because doing so can reduce handling and media consumption,especially in a multi-transfection protocol. In a preferred embodiment,the medium is prepared by first treating a concentrated solution ofserum albumin by contacting the concentrated solution of serum albuminwith one or more ion-exchange resins, then removing the one or moreion-exchange resins from the concentrated solution of serum albumin, andfinally adding the treated concentrated solution of serum albumin to theother components of the medium. In a more preferred embodiment, theconcentrated solution of serum albumin is further contacted withactivated charcoal before adding the concentrated solution of serumalbumin to the other components of the medium. In an even more preferredembodiment, the concentrated solution of serum albumin is firstcontacted with sodium octanoate, then raised to a temperature of atleast 50 C for at least 10 minutes, then contacted with one or moreion-exchange resins, then contacted with activated charcoal, then addedto the other components of the medium.

In certain situations, it is desirable to replace animal-derivedcomponents with non-animal-derived and/or recombinant components in partbecause non-animal-derived and/or recombinant components can be producedwith a higher degree of consistency than animal-derived components, andin part because non-animal-derived and/or recombinant components carryless risk of contamination with toxic and/or pathogenic substances thando animal-derived components. Certain embodiments are therefore directedto a protein that is non-animal-derived and/or recombinant. Otherembodiments are directed to a medium, wherein some or all of thecomponents of the medium are non-animal-derived and/or recombinant. In apreferred embodiment, the protein is recombinant serum albumin. Inanother preferred embodiment, the protein is recombinant human serumalbumin. In a more preferred embodiment, the protein is recombinantserum albumin and all of the components of the medium arenon-animal-derived and/or recombinant.

The N-terminus of serum albumin contains a nickel- and copper-bindingdomain, which is an important antigenic determinant Deleting theaspartic acid residue from the N-terminus of serum albumin eliminatesthe nickel- and copper-binding activity of serum albumin, and results ina hypoallergenic variant of the protein. Certain embodiments aretherefore directed to a protein that has modified bindingcharacteristics and/or other desirable characteristics such ashypoallergenicity. In a preferred embodiment, the protein is serumalbumin, and the serum albumin lacks the N-terminal aspartic acid ofserum albumin.

Other embodiments are directed to methods for transfecting a cell usingthe medium of the present invention. In a preferred embodiment, a cellis transfected with one or more nucleic acids, and the transfection isperformed using a transfection reagent, more preferably a lipid-basedtransfection reagent. In a more preferred embodiment, the one or morenucleic acids includes at least one RNA molecule. In another preferredembodiment, the cell is transfected with one or more nucleic acids, andthe one or more nucleic acids encodes at least one element of the groupcomprising: p53, Tert, a cytokine, a secreted protein, a membrane-boundprotein, an enzyme, a chromatin-modifying protein, a DNA-bindingprotein, a histone deacetylase, a pathogen-associated molecular pattern,and a tumor-associated antigen or a biologically active fragment,analogue, variant or family-member thereof. In another preferredembodiment, the cell is transfected repeatedly, preferably at least 2times during 10 consecutive days, more preferably at least 3 timesduring 7 consecutive days, even more preferably at least 4 times during6 consecutive days.

Reprogramming can be performed by transfecting cells with one or morenucleic acids encoding one or more reprogramming factors, and culturingthe cells in a medium that supports the reprogrammed cells. Examples ofreprogramming factors include, but are not limited to: Oct4 protein,Sox2 protein, Klf4 protein, c-Myc protein, 1-Myc protein, Tert protein,Nanog protein, Lin28 protein, Utf1 protein, Aicda protein, miR200micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA andbiologically active fragments, analogues, variants and family-membersthereof. It has now been discovered that the efficiency, speed, andreliability of reprogramming can be enhanced by using a medium thatsupports both the cells and the reprogrammed cells. Certain embodimentsare therefore directed to a medium that supports the growth of multiplecell types. In a preferred embodiment, the medium supports the growth ofhuman fibroblasts and human pluripotent stem cells. It has now beenfurther discovered that the efficiency, speed, and reliability ofreprogramming can be dramatically enhanced by transfecting cellsaccording to the methods of the present invention (see Examples).Certain embodiments are therefore directed to methods for reprogramminga cell by transfecting the cell according to the methods of the presentinvention. In a preferred embodiment, the cell is transfected with oneor more nucleic acids. In a more preferred embodiment, the one or morenucleic acids includes RNA molecules encoding Oct4 protein. In an evenmore preferred embodiment, the one or more nucleic acids includes RNAmolecules encoding Oct4, Sox2, Klf4, and c-Myc proteins. In a still morepreferred embodiment, the one or more nucleic acids also includes RNAmolecules encoding Lin28 protein. In another even more preferredembodiment, the RNA molecules also comprise at least one pseudouridineor 5-methylcytidine residue. In a still more preferred embodiment, atleast 20% of the uridine and cytidine residues of the RNA molecules arereplaced with pseudouridine and 5-methylcytidine residues, respectively.In a still more preferred embodiment, about 100% of the uridine andcytidine residues of the RNA molecules are replaced with pseudouridineand 5-methylcytidine residues, respectively. In one embodiment, the cellis a human skin cell, and the human skin cell is reprogrammed to apluripotent stem cell. In another embodiment, the cell is a human skincell, and the human skin cell is reprogrammed to a glucose-responsiveinsulin-producing cell. Examples of other cells that can be reprogrammedaccording to the methods of the present invention and other cells towhich a cell can be reprogrammed according to the methods of the presentinvention include, but are not limited to: skin cells, pluripotent stemcells, mesenchymal stem cells, β-cells, retinal pigmented epithelialcells, hematopoietic cells, cardiac cells, airway epithelial cells,neural stem cells, neurons, glial cells, bone cells, blood cells, anddental pulp stem cells.

Importantly, infecting skin cells with viruses encoding Oct4, Sox2,Klf4, and c-Myc, combined with culturing the cells in a medium thatsupports the growth of cardiomyocytes, has been shown to causereprogramming of the skin cells to cardiomyocytes, without firstreprogramming the skin cells to pluripotent stem cells¹⁶. In certainsituations, for example when generating a personalized therapeutic,direct reprogramming (reprogramming one somatic cell to another somaticcell without first reprogramming the somatic cell to a pluripotent stemcell, also known as “transdifferentiation”) may be desirable, in partbecause culturing pluripotent stem cells can be time-consuming andexpensive, the additional handling involved in establishing andcharacterizing a stable pluripotent stem cell line carries an increasedrisk of contamination, and the additional time in culture associatedwith first producing pluripotent stem cells carries an increased risk ofgenomic instability and the acquisition of mutations, including pointmutations, copy-number variations, and karyotypic abnormalities. Certainembodiments are therefore directed to methods for reprogramming asomatic cell, wherein the cell is reprogrammed to a somatic cell, andwherein a characterized pluripotent stem-cell line is not produced. In apreferred embodiment, the somatic cell is contacted with one or more RNAmolecules that encodes at least one protein selected from the groupcomprising: Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28, Utf1, and Aicda. Inanother preferred embodiment, the somatic cell is reprogrammed to asomatic cell selected from the group comprising: a skin cell, amesenchymal stem cell, a β-cell, a retinal pigmented epithelial cell, ahematopoietic cell, a cardiac cell, an airway epithelial cell, a neuralstem cell, a neuron, a glial cell, a bone cell, a blood cell, and adental pulp stem cell.

All previously reported methods for reprogramming cells by transfectingthem with RNA encoding reprogramming factors require the use of feeders.In many situations, the use of feeders is not desirable in part becausefeeders are generally derived from animal or allogeneic sources, andthus carry risks of immunogenicity and contamination with pathogens. Ithas now been discovered that the medium of the present invention canenable RNA reprogramming without feeders (see Examples). It has beenfurther discovered that reprogramming cells according to the methods ofthe present invention, wherein the cells are not contacted with feeders,can be rapid, efficient, and reliable. Certain embodiments are thereforedirected to methods for reprogramming a cell, wherein the cell is notcontacted with feeders. In a preferred embodiment, the cell istransfected with one or more RNA molecules encoding one or morereprogramming factors.

It has been further discovered that the starting cell density cancorrelate with reprogramming efficiency when cells are reprogrammedaccording to the methods of the present invention. Certain embodimentsare therefore directed to methods for reprogramming cells, wherein thecells are plated at a density of between 100 cells/cm² and 100,000cells/cm². In a preferred embodiment, the cells are plated at a densityof between 2000 cells/cm² and 20,000 cells/cm².

It has been further discovered that, in certain situations, fewer totaltransfections are required to reprogram a cell according to the methodsof the present invention than according to other methods (see Examples).Certain embodiments are therefore directed to methods for reprogramminga cell, wherein between 2 and 12 transfections are performed during 20consecutive days. In a preferred embodiment, between 4 and 10transfections are performed during 15 consecutive days. In a morepreferred embodiment, between 4 and 8 transfections are performed during10 consecutive days. It is recognized that when nucleic acids are addedto a medium in which a cell is cultured, the cell may likely come intocontact with and/or internalize more than one nucleic acid eithersimultaneously or at different times. A cell can therefore be contactedwith a nucleic acid more than once, i.e. repeatedly, even when nucleicacids are added only once to a medium in which the cell is cultured.

When cells are transfected and are cultured in the presence of feeders,the feeders are, in general, also transfected with the nucleic acid. Ithas now been discovered that feeders can prevent excessive transfectionof cells in part by removing excess transfection reagent-nucleic acidcomplexes from the medium. It has been further discovered that theefficiency, speed, and reliability of reprogramming without feeders canbe increased when the excess transfection reagent-nucleic acid complexesare rinsed off of the cells after transfection. Certain embodiments aretherefore directed to methods for transfecting a cell without usingfeeders, wherein the cell is rinsed after transfection. It is recognizedthat the rinsing can be performed using, for example, cell-culturemedium, transfection medium, basal medium, or a simple solution such asphosphate-buffered saline. In a preferred embodiment, the transfectionis repeated, and the cell is rinsed after each transfection. In a morepreferred embodiment, the transfection is repeated, the cell is rinsedafter each transfection, and the cell is reprogrammed.

Feeders can promote adhesion of cells to a surface by secretingmolecules such as collagen that bind to the surface (“cell-adhesionmolecules”). Proteins, including integrins, on the surface of cells canbind to these molecules, and cause the cells to adhere to the surface.It has now been discovered that cells can be reprogrammed withoutfeeders by coating a surface with one or more cell-adhesion molecules,and that fibronectin and vitronectin are particularly suited for thispurpose (see Examples). Certain embodiments are therefore directed tomethods for transfecting and/or reprogramming a cell, wherein the cellis contacted with a surface that is contacted with one or morecell-adhesion molecules. In a preferred embodiment, the one or morecell-adhesion molecules includes at least one element of the groupcomprising: poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin,vitronectin, collagen, and laminin or a biologically active fragment,analogue, variant or family-member thereof. In another preferredembodiment, the one or more cell-adhesion molecules is fibronectin or abiologically active fragment thereof. In yet another preferredembodiment, the fibronectin is recombinant. In a more preferredembodiment, the one or more cell-adhesion molecules is a mixture offibronectin and vitronectin or biologically active fragments thereof. Inan even more preferred embodiment, both the fibronectin and vitronectinare recombinant. It is recognized that the contacting of the surfacewith the one or more cell-adhesion molecules can be performedseparately, and/or by mixing the one or more cell-adhesion moleculeswith the medium.

Of note, nucleic acids can contain one or more non-canonical, or“modified”, residues (a residue other than adenine, guanine, thymine,uracil, and cytosine or the standard nucleoside, nucleotide,deoxynucleoside or deoxynucleotide derivatives thereof). Of particularnote, pseudouridine-triphosphate can be substituted foruridine-triphosphate in an in vitro-transcription reaction to yieldsynthetic RNA, wherein up to 100% of the uridine residues of thesynthetic RNA are replaced with pseudouridine residues¹⁷⁻²¹. Invitro-transcription yields RNA with residualimmunogenicityl^(12, 13, 22), even when pseudouridine and5-methylcytidine are completely substituted for uridine and cytidine,respectively¹⁵. For this reason, it is common to add animmunosuppressant to the transfection medium when transfecting cellswith RNA. In certain situations, adding an immunosuppressant to thetransfection medium is not desirable in part because the recombinantimmunosuppressant most commonly used for this purpose, B18R, isexpensive and difficult to manufacture. It has now been discovered thatcells can be reprogrammed according to the methods of the presentinvention, without using B18R or any other immunosuppressant (seeExamples). It has been further discovered that reprogramming cellsaccording to the methods of the present invention without usingimmunosuppressants can be rapid, efficient, and reliable (see Examples).Certain embodiments are therefore directed to methods for reprogramminga cell, wherein the transfection medium does not contain animmunosuppressant. In certain situations, such as when using a high celldensity, it is beneficial to add an immunosuppressant to the medium ofthe present invention. Certain embodiments are therefore directed tomethods for reprogramming, wherein the medium contains animmunosuppressant such as B18R or dexamethasone. In a preferredembodiment, cells are plated at less than 20,000 cells/cm², and thetransfection medium does not contain an immunosuppressant.

It has now been discovered that when an immunosuppressant is not used,reprogramming efficiency, speed, and reliability can be increased byreducing the nucleic-acid dose (see Examples), increasing the timebetween transfections, performing multiple media changes to removesecreted inflammatory cytokines from the medium, and/or performing oneor more rinsing steps to remove excess transfection reagent-nucleic acidcomplexes and/or inflammatory cytokines from the cells. Certainembodiments are therefore directed to methods for reprogramming a cell,wherein the transfection medium does not contain an immunosuppressant,and wherein the nucleic-acid dose is chosen to prevent excessivetoxicity. In a preferred embodiment, the nucleic-acid dose is less than2 μg/well of a 6-well plate, preferably between 0.25 μg/well of a 6-wellplate and 1 μg/well of a 6-well plate. Other embodiments are directed tomethods for reprogramming a cell, wherein the transfection medium doesnot contain an immunosuppressant, and wherein at least 2 transfectionsare performed, and the time between 2 transfections is at least 36hours. In a preferred embodiment, at least 5 transfections areperformed, and the time between each transfection is 48 hours. Otherembodiments are directed to methods for reprogramming a cell, whereinthe transfection medium does not contain an immunosuppressant, andwherein at least 2 media changes are performed between transfections.Other embodiments are directed to methods for reprogramming cells,wherein the transfection medium does not contain an immunosuppressant,and wherein the cells are rinsed after each transfection. In a preferredembodiment, the cells are rinsed with basal medium. In a more preferredembodiment, the cells are rinsed with transfection medium.

It has now been discovered that, in certain situations, certain specificmorphological changes, such as the formation of high-phase regions inthe cellular cytoplasm and clear, circular areas resembling thelipid-containing vesicles of adipocytes, are observed in cells that aretransfected according to the methods of the present invention. Examplesof these morphological changes are shown in the figures of theaccompanying drawings. It has been discovered that these morphologicalchanges can be predictive of transfection efficiency, toxicity, andreprogramming efficiency. Certain embodiments are therefore directed tomethods for developing a transfection protocol based on themorphological changes observed in cells after transfection with anucleic acid. Other embodiments are directed to methods for developing areprogramming protocol based on the morphological changes observed incells after transfection with a nucleic acid.

Reprogrammed cells produced according to certain preferred embodimentsof the present invention are suitable in particular for therapeuticapplications including transplantation into patients, as they do notcontain exogenous DNA sequences, and they are not exposed toanimal-derived or human-derived products, which are undefined, and whichmay contain toxic and/or pathogenic contaminants. Furthermore, the highspeed, efficiency, and reliability of the methods of the presentinvention reduce the risk of acquisition and accumulation of mutationsand other chromosomal abnormalities. Certain embodiments of the presentinvention can thus be used to generate cells that have a safety profilethat supports their use in therapeutic applications. For example,reprogramming cells using RNA and the medium of the present invention,wherein the medium does not contain animal or human-derived components,can yield cells that have not been exposed to allogeneic material.Certain embodiments are therefore directed to a reprogrammed cell thathas a desirable safety profile. In a preferred embodiment, thereprogrammed cell has a normal karyotype and fewer than 100 singlenucleotide variants in coding regions relative to the patient genome,more preferably fewer than 50 single nucleotide variants in codingregions relative to the patient genome, more preferably fewer than 10single nucleotide variants in coding regions relative to the patientgenome.

Certain embodiments are directed to a kit containing one or morematerials needed to practice the present invention. In a preferredembodiment, the kit contains the medium of the present invention and asolution containing RNA encoding one or more reprogramming factors. Inanother preferred embodiment, the kit further contains a transfectionreagent. In yet another preferred embodiment, the kit contains aliquotsof the medium of the present invention, wherein each aliquot containstransfection reagent-nucleic acid complexes that are stabilized eitherby chemical treatment or by freezing.

Endotoxins and nucleases can co-purify and/or become associated withother proteins, such as serum albumin. Recombinant proteins, inparticular, often have high levels of associated endotoxins andnucleases, due in part to the lysis of cells that can take place duringtheir production. Endotoxins and nucleases can be reduced, removed,replaced or otherwise inactivated by many of the methods of the presentinvention, including, for example, by acetylation, by addition of astabilizer such as sodium octanoate, followed by heat treatment, by theaddition of nuclease inhibitors to the albumin solution and/or medium,by crystallization, by contacting with one or more ion-exchange resins,by contacting with charcoal, by preparative electrophoresis or byaffinity chromatography. It has now been discovered that partially orcompletely reducing, removing, replacing or otherwise inactivatingendotoxins and/or nucleases from a medium and/or from one or morecomponents of a medium can increase the efficiency with which cells canbe transfected with nucleic acids, and the efficiency with which cellscan be reprogrammed. Certain embodiments are therefore directed to amethod for transfecting a cell with one or more nucleic acids, whereinthe transfection medium is treated to partially or completely reduce,remove, replace or otherwise inactivate one or more endotoxins and/ornucleases. In a preferred embodiment, the medium contains serum albumin,preferably recombinant serum albumin. In a more preferred embodiment,the serum albumin is contacted with sodium octanoate, and the serumalbumin is incubated at an elevated temperature, preferably at least 50C for at least 10 minutes. In an even more preferred embodiment, theserum albumin is further contacted with one or more ion-exchange resinsand/or charcoal. Other embodiments are directed to a medium that causesminimal degradation of nucleic acids. In a preferred embodiment, themedium contains less than 1 EU/mL, preferably less than 0.1 EU/mL, morepreferably less than 0.01 EU/mL. In another preferred embodiment, thecell-culture medium supports reprogramming.

It is recognized that protein-based lipid carriers such as serum albumincan be replaced with non-protein-based lipid carriers such asmethyl-beta-cyclodextrin. It is also recognized that the medium of thepresent invention can be used without a lipid carrier, whereintransfection is performed using a method that does not require thepresence of a lipid carrier, for example, using one or morepolymer-based transfection reagents or peptide-based transfectionreagents.

It is further recognized that many protein-associated molecules, such asmetals, can be highly toxic to cells. This toxicity can cause decreasedviability in culture, as well as the acquisition of mutations. Certainembodiments of the present invention thus have the additional benefit ofproducing cells that are free from toxic molecules.

The associated-molecule component of a protein can be measured bysuspending the protein in solution and measuring the conductivity of thesolution. Certain embodiments are therefore directed to a medium thatcontains a protein, wherein a 10% solution of the protein in water has aconductivity of less than 500 μmho/cm. In a preferred embodiment, thesolution has a conductivity of less than 50 μmho/cm.

A low-oxygen environment can be beneficial for the culture of many typesof cells. Certain embodiments are therefore directed to methods forculturing, transfecting and/or reprogramming cells according to themethods of the present invention, wherein the cells are cultured,transfected, and/or reprogrammed in a low-oxygen environment. In apreferred embodiment, the cells are cultured, transfected, and/orreprogrammed in an environment containing between 2% and 10% oxygen. Ina more preferred embodiment, the cells are cultured, transfected, and/orreprogrammed in an environment containing between 4% and 6% oxygen.

The amount of nucleic acid delivered to cells can be increased toincrease the desired effect of the nucleic acid. However, increasing theamount of nucleic acid delivered to cells beyond a certain point causesa decrease in the viability of the cells, due in part to toxicity of thetransfection reagent. It has now been discovered that when a nucleicacid is delivered to a population of cells in a fixed volume (forexample, cells in a region of tissue or cells grown in a cell-culturevessel), the amount of nucleic acid delivered to each cell depends onthe total amount of nucleic acid delivered to the population of cellsand to the density of the cells, with a higher cell density resulting inless nucleic acid being delivered to each cell. In certain embodimentsof the present invention, a cell is transfected with one or more nucleicacids more than once. Under certain conditions, for example when thecells are proliferating, the cell density may change from onetransfection to the next. Certain embodiments are therefore directed tomethods for transfecting a cell with a nucleic acid, wherein the cell istransfected more than once, and wherein the amount of nucleic aciddelivered to the cell is different for two subsequent transfections. Ina preferred embodiment, the cell proliferates between two of thetransfections, and the amount of nucleic acid delivered to the cell isgreater for the second of the two transfections than for the first ofthe two transfections. In another preferred embodiment, the cell istransfected more than twice, and the amount of nucleic acid delivered tothe cell is greater for the second of three transfections than for thefirst of the same three transfections, and the amount of nucleic aciddelivered to the cells is greater for the third of the same threetransfections than for the second of the same three transfections. Inyet another preferred embodiment, the cell is transfected more thanonce, and the maximum amount of nucleic acid delivered to the cellduring each transfection is sufficiently low to yield at least 80%viability (i.e. at least 80% of the cells survive each transfection) forat least two consecutive transfections.

It has now been further discovered that modulating the amount of nucleicacid delivered to a population of proliferating cells in a series oftransfections can result in both an increased effect of the nucleic acidand increased viability of the cells. It has also now been discoveredthat, in certain situations, when cells are contacted with one or morenucleic acids encoding one or more reprogramming factors in a series oftransfections, the efficiency of reprogramming is increased when theamount of nucleic acid delivered in later transfections is greater thanthe amount of nucleic acid delivered in earlier transfections, for atleast part of the series of transfections. Certain embodiments aretherefore directed to a method for reprogramming a cell, wherein one ormore nucleic acids is repeatedly delivered to the cell in a series oftransfections, and the amount of the nucleic acid delivered to the cellis greater for at least one later transfection than for at least oneearlier transfection. In a preferred embodiment, the cell is transfectedbetween 2 and 10 times, preferably between 3 and 8 times, morepreferably between 4 and 6 times. In another preferred embodiment, theone or more nucleic acids includes at least one RNA molecule, the cellis transfected between 2 and 10 times, and the amount of nucleic aciddelivered to the cell in each transfection is the same as or greaterthan the amount of nucleic acid delivered to the cell in the most recentprevious transfection. In yet another preferred embodiment, the amountof nucleic acid delivered to the cell in the first transfection isbetween 20 ng/cm² and 250 ng/cm². In yet another preferred embodiment,the amount of nucleic acid delivered to the cell in the lasttransfection is between 100 ng/cm² and 600 ng/cm². In yet anotherpreferred embodiment, the cell is transfected 5 times at intervals ofbetween 12 and 48 hours, and the amount of nucleic acid delivered to thecell is 25 ng/cm² for the first transfection, 50 ng/cm² for the secondtransfection, 100 ng/cm² for the third transfection, 200 ng/cm² for thefourth transfection, and 400 ng/cm² for the fifth transfection.

In certain situations, the performance of a medium can be improved byconditioning the medium. It has now been discovered that thetransfection efficiency and viability of cells cultured in the medium ofthe present invention can be improved by conditioning the medium.Certain embodiments are therefore directed to a method for conditioninga medium. Other embodiments are directed to a medium that isconditioned. In a preferred embodiment, the feeders are fibroblasts, andthe medium is conditioned for approximately 24 hours. Other embodimentsare directed to a method for transfecting a cell, wherein thetransfection medium is conditioned. Other embodiments are directed to amethod for reprogramming a cell, wherein the medium is conditioned. In apreferred embodiment, the feeders are mitotically inactivated, forexample, by exposure to a chemical such as mitomycin-C or by exposure togamma radiation. In certain situations, it is beneficial to use onlyautologous materials, in part to avoid the risk of disease transmissionfrom the feeders to the cell being reprogrammed. Certain embodiments aretherefore directed to a method for transfecting a cell, wherein thetransfection medium is conditioned, and wherein the feeders are derivedfrom the same individual as the cell being transfected. Otherembodiments are directed to a method for reprogramming a cell, whereinthe medium is conditioned, and wherein the feeders are derived from thesame individual as the cell being reprogrammed.

Several molecules are added to media by conditioning. Certainembodiments are therefore directed to a medium that is supplemented withone or more molecules that are present in a conditioned medium. In apreferred embodiment, the medium is supplemented with Wnt1, Wnt2, Wnt3,Wnt3a or a biologically active fragment, analogue, variant, agonist, orfamily-member thereof. In another preferred embodiment, the medium issupplemented with TGF-β or a biologically active fragment, analogue,variant, agonist, or family-member thereof. In yet another preferredembodiment, a cell is reprogrammed according to the method of thepresent invention, wherein the medium is not supplemented with TGF-β forbetween 1 and 5 days, and is then supplemented with TGF-β for at least 2days. In yet another preferred embodiment, the medium is supplementedwith IL-6, IL-6R or a biologically active fragment, analogue, variant,agonist, or family-member thereof. In yet another preferred embodiment,the medium is supplemented with a sphingolipid or a fatty acid. In yetanother preferred embodiment, the sphingolipid is lysophosphatidic acid,lysosphingomyelin, sphingosine-1-phosphate or a biologically activeanalogue, variant or derivative thereof.

In addition to mitotically inactivating cells, under certain conditions,irradiation can change the gene expression of cells, causing cells toproduce less of certain proteins and more of certain other proteins thatnon-irradiated cells, for example, members of the Wnt family ofproteins^(23, 24). In addition, certain members of the Wnt family ofproteins promote the growth and transformation of cells^(25, 26). It hasnow been discovered that, in certain situations, the efficiency of RNAreprogramming can be greatly increased by contacting the cell with amedium that is conditioned using irradiated feeders instead ofmitomycin-c-treated feeders. It has been further discovered that theincrease in reprogramming efficiency observed when using irradiatedfeeders is caused in part by Wnt proteins that are secreted by thefeeders. Certain embodiments are therefore directed to a method forreprogramming a cell, wherein the cell is contacted with Wnt1, Wnt2,Wnt3, Wnt3a or a biologically active fragment, analogue, variant,family-member or agonist thereof, including agonists of downstreamtargets of Wnt proteins, and/or agents that mimic one or more of thebiological effects of Wnt proteins, for example,2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine²⁷.In a preferred embodiment, the cell is further contacted with one ormore RNA molecules. In a more preferred embodiment, at least 2transfections are performed during 5 consecutive days. In anotherpreferred embodiment, the cell is a human skin cell. In yet anotherpreferred embodiment, the one or more RNA molecules encodes the proteinsOct4, Sox2, Klf4, and c-Myc.

It is recognized that the medium of the present invention can be used tomaintain cells, including fibroblasts and human pluripotent stem cells,in culture (i.e. as a “maintenance medium”; see Examples). Certainembodiments are therefore directed to the medium of the presentinvention, wherein the medium is used as a maintenance medium. In apreferred embodiment, the medium does not contain any human-derivedcomponents. In a more preferred embodiment, the medium is chemicallydefined. It is also recognized that the addition of components that areknown to promote the growth of certain cell types, for examplelipid-rich albumin, which can promote the growth of pluripotent stemcells⁴, can be added to the medium of the present invention to promotethe growth of these cell types when the medium is used as a transfectionmedium, as a maintenance medium, or as both a transfection medium and amaintenance medium.

DNA-based reprogramming methods generally use cells that are derivedfrom established cultures of primary cells, such as fibroblasts. Becauseof the low efficiency of these methods, DNA-based reprogramming isdifficult or impossible to use with cells derived from patient samples,which generally contain too few cells to produce a sufficient number ofreprogrammed cells using these methods. In contrast, the high efficiencyof certain embodiments of the present invention allows reliablereprogramming from small numbers of cells, including from single cells.Certain embodiments of the present invention can thus be used toreprogram cells from a biopsy sample, without first establishing a largeculture (see Examples). Reprogramming cells directly from a biopsy isdesirable in certain situations, especially when generating apersonalized therapeutic, in part because establishing a large cultureof primary cells is time-consuming, the additional handling involved inestablishing a large culture carries an increased risk of contamination,and the additional time in culture carries an increased risk of genomicinstability and the acquisition of mutations, including point mutations,copy-number variations, and karyotypic abnormalities. Certainembodiments are therefore directed to methods for reprogramming a cellby harvesting the cell from a patient or from a biopsy sample, andreprogramming the cell according to the methods of the presentinvention. In a preferred embodiment, the cell is reprogrammed withoutfirst establishing a large culture (i.e. a first transfection isperformed before the culture is passaged more than twice). In anotherpreferred embodiment, the cell is harvested from a patient, and a firsttransfection is performed after no more than 14 days from the time thecell is first plated. In yet another preferred embodiment, the cell isharvested from a biopsy sample, and a first transfection is performedafter no more than 7 days from the time the cell is first plated. In yetanother preferred embodiment, the biopsy is a full-thickness dermalpunch biopsy, the cell is harvested from the biopsy sample by treatmentwith one or more enzymes, the cell is plated on a surface that is coatedwith a cell-adhesion molecule and/or the cell is plated in a medium thatcontains a cell-adhesion molecule, the cell is transfected with one ormore nucleic acids comprising at least one RNA molecule, and a firsttransfection is performed after no more than 14 days from the time thecell is first plated. In yet another preferred embodiment, the cell isharvested from blood. In yet another preferred embodiment, the cell isplated in a medium containing one or more proteins that is derived fromthe patient's blood. In yet another preferred embodiment, the cell isplated in DMEM/F12+2 mM L-alanyl-L-glutamine+between 5% and 25%patient-derived serum, more preferably between about 10% and about 20%patient-derived serum, most preferably about 20% patient-derived serum.

It has now been discovered that cells, including fibroblasts, can beefficiently isolated from tissue by placing the tissue in a container,and suspending the tissue at the air-liquid interface of a solutioncontaining an enzyme that digests one or more protein components of thetissue, such that the cells that are freed from the tissue fall to thebottom of the container, where they can then be collected. This methodis advantageous, in part because it enables isolation and collection ofcells from a tissue without mechanical disruption of the tissue, whichcan damage cells and create a large amount of debris. It has now beenfurther discovered that the lipophilic nature of the epidermisfacilitates suspension of skin tissue at an air-liquid interface, andthat fibroblasts are efficiently isolated from tissue obtained by afull-thickness skin punch biopsy using this method. Certain embodimentsare therefore directed to a method for isolating cells from a tissuesample by suspending the tissue sample at an air-liquid interface,wherein the liquid contains an enzyme that digests one or more proteincomponents of the tissue. In a preferred embodiment, the tissue sampleis a full-thickness skin punch biopsy sample. In another preferredembodiment, the enzyme is collagenase. In a more preferred embodiment,the collagenase is animal-component free. In yet another preferredembodiment, the collagenase is present at a concentration of between 0.1mg/mL and 10 mg/mL, more preferably between 0.5 mg/mL and 5 mg/mL. Inyet another preferred embodiment, the tissue is suspended for between 1h and 24 h, more preferably between 6 h and 12 h. This method is alsoadvantageous as a method for isolating cells for reprogramming, in partbecause it can yield a well-dissociated cell suspension without the needfor harsh mechanical disruption, and because it can eliminate the needfor establishing a stable culture before transfection.

It has now been further discovered that, in certain situations,transfecting cells with a mixture of RNA encoding Oct4, Sox2, Klf4, andc-Myc using the medium of the present invention causes the rate ofproliferation of the cells to increase. When the amount of RNA deliveredto the cells is too low to ensure that all of the cells are transfected,only a fraction of the cells may show an increased proliferation rate.In certain situations, such as when generating a personalizedtherapeutic, increasing the proliferation rate of cells may bedesirable, in part because doing so can reduce the time necessary togenerate the therapeutic, and therefore can reduce the cost of thetherapeutic. Certain embodiments are therefore directed to transfectinga cell with a mixture of RNA encoding Oct4, Sox2, Klf4, and c-Myc usingthe medium of the present invention, wherein the cell shows an increasedproliferation rate. In a preferred embodiment, the cell is harvestedfrom a biopsy sample, and the cell is transfected between 2 and 7 times.In another preferred embodiment, cells showing an increasedproliferation rate are isolated from the culture. In yet anotherpreferred embodiment, cells showing an increased proliferation rate areexpanded and cultured in a medium that supports the growth of one ormore cell types, and are reprogrammed to a cell of one of the one ormore cell types.

Certain embodiments are directed to therapeutics comprising one or morecells that are transfected and/or reprogrammed according to the methodsof the present invention. In a preferred embodiment, a cell istransfected and/or reprogrammed, and the transfected and/or reprogrammedcell is introduced to a patient. In another preferred embodiment, thecell is harvested from the same patient to whom the transfected and/orreprogrammed cell is introduced. Examples of diseases that can betreated with therapeutics of the present invention include, but are notlimited to: Alzheimer's disease, spinal cord injury, amyotrophic lateralsclerosis, cystic fibrosis, heart disease, including ischemic anddilated cardiomyopathy, macular degeneration, Parkinson's disease,Huntington's disease, diabetes, sickle-cell anemia, thalassemia, Fanconianemia, xeroderma pigmentosum, muscular dystrophy, severe combinedimmunodeficiency, hereditary sensory neuropathy, and HIV/AIDS. Incertain embodiments, the therapeutic comprises a cosmetic. In apreferred embodiment, a cell is harvested from a patient, the cell isreprogrammed and expanded to a large number of adipose cells, thusproducing a cosmetic, and the cosmetic is introduced to the patient. Ina more preferred embodiment, the cosmetic is further used for tissuereconstruction.

While detailed examples are provided herein for the production ofspecific types of cells and for the production of therapeuticscomprising specific types of cells, it is recognized that the methods ofthe present invention can be used to produce many other types of cells,and to produce therapeutics comprising one or more of many other typesof cells, for example, by reprogramming a cell according to the methodsof the present invention, and culturing the cell under conditions thatmimic one or more aspects of development by providing conditions thatresemble the conditions present in the cellular microenvironment duringdevelopment.

Certain embodiments are directed to libraries of therapeutics comprisingcells with a variety of human leukocyte antigen (HLA) types(“HLA-matched libraries”). An HLA-matched library is beneficial in partbecause it provides for the rapid production and/or distribution oftherapeutics without the patient having to wait for a therapeutic to beproduced from the patient's cells. Such a library is particularlybeneficial for the treatment of heart disease and diseases of the bloodand/or immune system for which patients may benefit from the immediateavailability of a therapeutic.

Certain embodiments are directed to cells that are used as tissue/organmodels and/or disease models. In one embodiment, a skin cell isreprogrammed and expanded to a large number of cardiac cells, and thecardiac cells are used for screening bioactive molecules forcardiotoxicity (i.e. safety testing). In another embodiment, a skin cellfrom a patient with Alzheimer's disease is reprogrammed and expanded toa large number of cortical neurons, and the cortical neurons are usedfor screening bioactive molecules for reducing the accumulation ofinsoluble plaques (i.e. efficacy testing). Certain embodiments of thepresent invention are therefore useful for safety testing and/orefficacy testing.

Certain embodiments are directed to a method for encapsulating cellsand/or seeding cells in a scaffold, and to cells that are encapsulatedand/or cells that are seeded in a scaffold. In certain situations,encapsulating cells is beneficial in part because encapsulated cells canbe less immunogenic than non-encapsulated cells. In a preferredembodiment, a cell is reprogrammed to a glucose-responsiveinsulin-producing cell, the glucose-responsive insulin-producing cell isencapsulated in a material such as alginate, and the encapsulatedglucose-responsive insulin-producing cell is introduced into a patientwith type 1 diabetes. In another preferred embodiment, the introducingis by intraperitoneal injection or intraportal injection. In certainsituations, seeding cells in a scaffold is beneficial in part because ascaffold can provide mechanical stability. In a preferred embodiment, acell is reprogrammed and expanded into a large number of fibroblasts andkeratinocytes, the fibroblasts and keratinocytes are seeded in ascaffold comprising collagen, and the seeded scaffold is applied to awound, forming a synthetic skin graft. In another preferred embodiment,a cell is reprogrammed, the reprogrammed cell is mixed with a scaffoldin liquid or slurry form, the mixture is introduced into the patient,and the stiffness of the scaffold increases upon or after introduction.

Certain embodiments are directed to a method for purifying the cells ofthe present invention. Reprogramming often produces populations of cellsincluding cells with the desired phenotype and cells with one or moreundesired phenotypes. Certain embodiments are therefore directed to amethod for purifying reprogrammed cells. In a preferred embodiment, thecells are purified using a density gradient. In another preferredembodiment, the cells are purified by contacting the cells with one ormore antibodies that allows the separation of cells having one or moredesired phenotypes from cells having one or more undesired phenotypes.In a more preferred embodiment, the antibody is bound to a substrate,preferably a magnetic bead. In another more preferred embodiment, theantibody is bound to a fluorescent molecule, and the separation isperformed by fluorescence activated cell sorting (FACS) or other similarmeans. In another preferred embodiment, cells with an undesiredphenotype are prevented from proliferating, preferably by contacting thecells with one or more molecules that prevents the cells from dividing,preferably mitomycin-c, 5-aza-deoxycytidine, fluorouracil or abiologically active analogue or derivative thereof. Other embodimentsare directed to a therapeutic comprising cells that are purified toenrich the fraction of cells having one or more desired phenotypes.

Certain embodiments are directed to a method for producing animalmodels, including models of mutations and diseases. In one embodiment,an animal skin cell is reprogrammed to a pluripotent stem cell accordingto the methods of the present invention. In another embodiment, 1-100reprogrammed cells are injected into a blastocyst, and the blastocyst isimplanted into the uterine horn of an animal. In a preferred embodiment,the animal is selected from the group comprising: a cat, a dog, a mouse,a pig, a primate, and a rat.

The present invention therefore has the aim of providing products forboth research and therapeutic use.

EXAMPLES Example 1 Synthesis of Reprogramming RNA

RNA encoding the human proteins Oct4, Sox2, Klf4, c-Myc-2 (T58A), andLin28 and comprising adenosine, guanosine, pseudouridine, and5-methylcytidine residues¹⁷⁻²⁰, was synthesized from DNA templates usingthe T7 High Yield RNA Synthesis Kit (New England Biolabs, Inc.),according to the manufacturer's instructions. The resulting RNA wasanalyzed by agarose gel electrophoresis to assess the quality of the RNA(FIG. 1). The RNA was then diluted to 200 ng/μL, and an RNase inhibitor(Superase-In™, Life Technologies Corporation) was added at aconcentration of 1 μL/100 μg of RNA. RNA solutions were stored at 4 Cindefinitely. RNA encoding Oct4, Sox2, Klf4, c-Myc-2 (T58A), and Lin28was mixed at a molar ratio of 3:1:1:1:1.

Example 2 Transfection of Cells with Reprogramming RNA

2 ug RNA and 6 μL transfection reagent (Lipofectamine™ RNAiMAX, LifeTechnologies Corporation) were first diluted separately in complexationmedium (Opti-MEM®, Life Technologies Corporation) to a total volume of60 μL each. Diluted RNA and transfection reagent were then mixed andincubated for 15 min at room temperature, according to the transfectionreagent-manufacturer's instructions. Complexes were then added to cellsin culture. Between 60 μL and 120 μL of complexes were added to eachwell of a 6-well plate, which already contained 2 mL of transfectionmedium per well. Plates were then shaken gently to distribute thecomplexes throughout the well. Cells were incubated with complexes for 2hours to overnight, before replacing the medium with fresh transfectionmedium (2 mL/well).

Example 3 Analysis of the Ability of Untreated Human Serum AlbuminPreparations to Support Nucleic Acid Transfection and RNA Reprogramming

Primary human neonatal fibroblasts were cultured in medium with orwithout 5 mg/mL HSA. Cohn Fraction V (A6784, Sigma-Aldrich Co. LLC.),and four different recombinant HSA preparations (A6608, A7736, A9731,and A9986, all from Sigma-Aldrich Co. LLC.) were screened. Cells weretransfected according to Example 2, with RNA synthesized according toExample 1. While untransfected cells grew well in media containing anyof the HSA preparations, in transfected wells, each of the HSApreparations yielded dramatically different cell morphologies and celldensities, and none resulted in morphological changes indicative ofreprogramming (FIG. 2).

Example 4 Production of Octanoate-Treated Human Serum Albumin

A 10% solution of HSA was pre-incubated with 22 mM sodium chloride and16 mM sodium octanoate (Sigma-Aldrich Co. LLC), and was incubated at 37C for 3 hours before assembly of the complete medium.

Example 5 Analysis of Transfection Efficiency and Viability of CellsCultured in Media Containing Octanoate-Treated Human Serum Albumin

Primary human neonatal fibroblasts were cultured in media containingrecombinant HSA treated according to Example 4 or containing treatedblood-derived HSA (Bio-Pure HSA, Biological Industries). Cells weretransfected daily, according to Example 2, with RNA synthesizedaccording to Example 1, beginning on day 0. Pictures were taken on day3. Several small areas of cells undergoing morphological changesresembling mesenchymal to epithelial transition were observed in thewells containing octanoate, indicating an increased transfectionefficiency (FIG. 3, second row, arrows). Many large areas ofmorphological changes resembling mesenchymal to epithelial transitionwere observed in the samples containing the treated blood-derived HSA(FIG. 3, fourth row, arrows). In both cases, the morphological changeswere characteristic of reprogramming.

Example 6 Reprogramming Human Fibroblasts Using Media ContainingOctanoate-Treated Human Serum Albumin

Primary human neonatal fibroblasts were plated in 6-well plates at adensity of 5000 cells/well in fibroblast medium (DMEM+10% fetal bovineserum). After 6 hours, the medium was replaced with transfection mediumcontaining octanoate-treated HSA. The cells were transfected daily,according to Example 2, with RNA synthesized according to Example 1,beginning on day 0. By day 5, the well contained several areas of cellsexhibiting morphology consistent with reprogramming. This experiment didnot include the use of feeders or immunosuppressants.

Example 7 Treatment of Human Serum Albumin Using Ion-ExchangeChromatography

A 20% solution of recombinant HSA produced in Pichia pastoris (A7736,Sigma-Aldrich Co. LLC.) was prepared by dissolving 2 g of HSA in 10 mLof nuclease-free water with gentle agitation at room temperature. TheHSA solution was then deionized by first adding 1 g of mixed-beddeionizing resin (AG 501-X8(D), Bio-Rad Laboratories, Inc.), and rockingfor 1 h at room temperature. The HSA solution was then decanted into atube containing 5 g of fresh resin, and was rocked for 4 h at roomtemperature. Finally, the deionized HSA solution was decanted, adjustedto a 10% total protein content with nuclease-free water,filter-sterilized using a 0.2 μm PES-membrane filter, and stored at 4 C.

Example 8 Transfection Medium Formulation

A cell-culture medium was developed to support efficient transfection ofcells with nucleic acids and efficient reprogramming:

DMEM/F12+10 μg/mL insulin+5.5 μg/mL transferrin+6.7 ng/mL sodiumselenite+20 ng/mL bFGF+5 mg/mL treated human serum albumin.

Variants of this medium were also developed to provide improvedperformance when used with specific transfection reagents, specificnucleic acids, and specific cell types:

DMEM/F12+10 μg/mL insulin+5.5 μg/mL transferrin+6.7 ng/mL sodiumselenite+4.5 μg/mL cholesterol+20 ng/mL bFGF+5 mg/mL treated human serumalbumin,

DMEM/F12+10 μg/mL insulin+5.5 μg/mL transferrin+6.7 ng/mL sodiumselenite+10 μg/mL fatty acids+20 ng/mL bFGF+5 mg/mL treated human serumalbumin,

DMEM/F12+10 μg/mL insulin+5.5 μg/mL transferrin+6.7 ng/mL sodiumselenite+4.5 μg/mL cholesterol+10 μg/mL fatty acids+20 ng/mL bFGF+5mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+10 μg/mL insulin+5.5 μg/mL transferrin+6.7 ng/mLsodium selenite+4.5 μg/mL cholesterol+10 μg/mL fatty acids+20 ng/mLbFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+4.5 μg/mL cholesterol+10μg/mL fatty acids+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+4.5 μg/mL cholesterol+10μg/mL cod liver oil fatty acids (methyl esters)+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherol acetate+1μg/mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1%Pluronic F-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+10μg/mL cod liver oil fatty acids (methyl esters)+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherol acetate+1μg/mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1%Pluronic F-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+25 μg/mL polyoxyethylenesorbitan monooleate+2 μg/mLD-alpha-tocopherol acetate+1 μg/mL L-ascorbic acid 2-phosphatesesquimagnesium salt hydrate+0.1% Pluronic F-68+20 ng/mL bFGF+5 mg/mLtreated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+10 μg/mL cod liver oil fatty acids (methyl esters)+2μg/mL D-alpha-tocopherol acetate+1 μg/mL L-ascorbic acid 2-phosphatesesquimagnesium salt hydrate+0.1% Pluronic F-68+20 ng/mL bFGF+5 mg/mLtreated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+10 μg/mL cod liver oil fatty acids (methyl esters)+25μg/mL polyoxyethylenesorbitan monooleate+1 μg/mL L-ascorbic acid2-phosphate sesquimagnesium salt hydrate+0.1% Pluronic F-68+20 ng/mLbFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+10 μg/mL cod liver oil fatty acids (methyl esters)+25μg/mL polyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+0.1% Pluronic F-68+20 ng/mL bFGF+5 mg/mL treated human serumalbumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+10 μg/mL cod liver oil fatty acids (methyl esters)+25μg/mL polyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1 μg/mL L-ascorbic acid 2-phosphate sesquimagnesium salthydrate+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+10 μg/mL cod liver oil fatty acids (methyl esters)+25μg/mL polyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1 μg/mL L-ascorbic acid 2-phosphate sesquimagnesium salthydrate+0.1% Pluronic F-68+20 ng/mL bFGF+5 mg/mL treated human serumalbumin*,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+0.5 μg/mL myristicacid+1.85 μg/mL palmitic acid+2.5 μg/mL stearic acid+0.25 μg/mLpalmitoleic acid+25 μg/mL polyoxyethylenesorbitan monooleate+2 μg/mLD-alpha-tocopherol acetate+1.85 μg/mL oleic acid+0.65 μg/mL linoleicacid+1 μg/mL L-ascorbic acid 2-phosphate sesquimagnesium salthydrate+0.1% Pluronic F-68+20 ng/mL bFGF+5 mg/mL treated human serumalbumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL gamma-linolenic acid+0.5 μg/mLdihomo-gamma-linolenic acid+0.5 μg/mL octadecatetraenoic acid+0.5 μg/mLeicosapentaenoic acid+0.5 μg/mL docosahexaenoic acid+0.5 μg/mLarachidonic acid+0.5 μg/mL myristic acid+1.85 μg/mL palmitic acid+2.5μg/mL stearic acid+0.25 μg/mL palmitoleic acid+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1.85 μg/mL oleic acid+0.65 μg/mL linoleic acid+1 μg/mLL-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1% PluronicF-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLdihomo-gamma-linolenic acid+0.5 μg/mL octadecatetraenoic acid+0.5 μg/mLeicosapentaenoic acid+0.5 μg/mL docosahexaenoic acid+0.5 μg/mLarachidonic acid+0.5 μg/mL myristic acid+1.85 μg/mL palmitic acid+2.5μg/mL stearic acid+0.25 μg/mL palmitoleic acid+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1.85 μg/mL oleic acid+0.65 μg/mL linoleic acid+1 μg/mLL-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1% PluronicF-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL octadecatetraenoic acid+0.5 μg/mLeicosapentaenoic acid+0.5 μg/mL docosahexaenoic acid+0.5 μg/mLarachidonic acid+0.5 μg/mL myristic acid+1.85 μg/mL palmitic acid+2.5μg/mL stearic acid+0.25 μg/mL palmitoleic acid+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1.85 μg/mL oleic acid+0.65 μg/mL linoleic acid+1 μg/mLL-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1% PluronicF-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLeicosapentaenoic acid+0.5 μg/mL docosahexaenoic acid+0.5 μg/mLarachidonic acid+0.5 μg/mL myristic acid+1.85 μg/mL palmitic acid+2.5μg/mL stearic acid+0.25 μg/mL palmitoleic acid+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1.85 μg/mL oleic acid+0.65 μg/mL linoleic acid+1 μg/mLL-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1% PluronicF-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL docosahexaenoic acid+0.5 μg/mLarachidonic acid+0.5 μg/mL myristic acid+1.85 μg/mL palmitic acid+2.5μg/mL stearic acid+0.25 μg/mL palmitoleic acid+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1.85 μg/mL oleic acid+0.65 μg/mL linoleic acid+1 μg/mLL-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1% PluronicF-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLarachidonic acid+0.5 μg/mL myristic acid+1.85 μg/mL palmitic acid+2.5μg/mL stearic acid+0.25 μg/mL palmitoleic acid+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1.85 μg/mL oleic acid+0.65 μg/mL linoleic acid+1 μg/mLL-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1% PluronicF-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL myristic acid+1.85 μg/mL palmiticacid+2.5 μg/mL stearic acid+0.25 μg/mL palmitoleic acid+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1.85 μg/mL oleic acid+0.65 μg/mL linoleic acid+1 μg/mLL-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1% PluronicF-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+1.85 μg/mL palmiticacid+2.5 μg/mL stearic acid+0.25 μg/mL palmitoleic acid+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1.85 μg/mL oleic acid+0.65 μg/mL linoleic acid+1 μg/mLL-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1% PluronicF-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+0.5 μg/mL myristicacid+2.5 μg/mL stearic acid+0.25 μg/mL palmitoleic acid+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1.85 μg/mL oleic acid+0.65 μg/mL linoleic acid+1 μg/mLL-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1% PluronicF-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+0.5 μg/mL myristicacid+1.85 μg/mL palmitic acid+0.25 μg/mL palmitoleic acid+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1.85 μg/mL oleic acid+0.65 μg/mL linoleic acid+1 μg/mLL-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1% PluronicF-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+0.5 μg/mL myristicacid+1.85 μg/mL palmitic acid+2.5 μg/mL stearic acid+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1.85 μg/mL oleic acid+0.65 μg/mL linoleic acid+1 μg/mLL-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1% PluronicF-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+0.5 μg/mL myristicacid+1.85 μg/mL palmitic acid+2.5 μg/mL stearic acid+0.25 μg/mLpalmitoleic acid+25 μg/mL polyoxyethylenesorbitan monooleate+2 μg/mLD-alpha-tocopherol acetate+0.65 μg/mL linoleic acid+1 μg/mL L-ascorbicacid 2-phosphate sesquimagnesium salt hydrate+0.1% Pluronic F-68+20ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+0.5 μg/mL myristicacid+1.85 μg/mL palmitic acid+2.5 μg/mL stearic acid+0.25 μg/mLpalmitoleic acid+25 μg/mL polyoxyethylenesorbitan monooleate+2 μg/mLD-alpha-tocopherol acetate+1.85 μg/mL oleic acid+1 μg/mL L-ascorbic acid2-phosphate sesquimagnesium salt hydrate+0.1% Pluronic F-68+20 ng/mLbFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+0.5μg/mL alpha-linolenic acid+0.5 μg/mL gamma-linolenic acid+0.5 μg/mLdihomo-gamma-linolenic acid+0.5 μg/mL octadecatetraenoic acid+0.5 μg/mLeicosapentaenoic acid+0.5 μg/mL docosahexaenoic acid+0.5 μg/mLarachidonic acid+0.5 μg/mL myristic acid+1.85 μg/mL palmitic acid+2.5μg/mL stearic acid+0.25 μg/mL palmitoleic acid+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1.85 μg/mL oleic acid+0.65 μg/mL linoleic acid+1 μg/mLL-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1% PluronicF-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+0.5 μg/mL myristicacid+1.85 μg/mL palmitic acid+2.5 μg/mL stearic acid+0.25 μg/mLpalmitoleic acid+2 μg/mL D-alpha-tocopherol acetate+1.85 μg/mL oleicacid+0.65 μg/mL linoleic acid+1 μg/mL L-ascorbic acid 2-phosphatesesquimagnesium salt hydrate+0.1% Pluronic F-68+20 ng/mL bFGF+5 mg/mLtreated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+0.5 μg/mL myristicacid+1.85 μg/mL palmitic acid+2.5 μg/mL stearic acid+0.25 μg/mLpalmitoleic acid+25 μg/mL polyoxyethylenesorbitan monooleate+1.85 μg/mLoleic acid+0.65 μg/mL linoleic acid+1 μg/mL L-ascorbic acid 2-phosphatesesquimagnesium salt hydrate+0.1% Pluronic F-68+20 ng/mL bFGF+5 mg/mLtreated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+0.5 μg/mL myristicacid+1.85 μg/mL palmitic acid+2.5 μg/mL stearic acid+0.25 μg/mLpalmitoleic acid+1.85 μg/mL oleic acid+0.65 μg/mL linoleic acid+1 μg/mLL-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+0.1% PluronicF-68+20 ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+0.5 μg/mL myristicacid+1.85 μg/mL palmitic acid+2.5 μg/mL stearic acid+0.25 μg/mLpalmitoleic acid+25 μg/mL polyoxyethylenesorbitan monooleate+2 μg/mLD-alpha-tocopherol acetate+1.85 μg/mL oleic acid+1 μg/mL L-ascorbic acid2-phosphate sesquimagnesium salt hydrate+0.1% Pluronic F-68+20 ng/mLbFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+0.5μg/mL alpha-linolenic acid+0.5 μg/mL gamma-linolenic acid+0.5 μg/mLdihomo-gamma-linolenic acid+0.5 μg/mL octadecatetraenoic acid+0.5 μg/mLeicosapentaenoic acid+0.5 μg/mL docosahexaenoic acid+0.5 μg/mLarachidonic acid+0.5 μg/mL myristic acid+1.85 μg/mL palmitic acid+2.5μg/mL stearic acid+0.25 μg/mL palmitoleic acid+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1.85 μg/mL oleic acid+1 μg/mL L-ascorbic acid 2-phosphatesesquimagnesium salt hydrate+0.1% Pluronic F-68+20 ng/mL bFGF+5 mg/mLtreated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL octadecatetraenoic acid+0.5 μg/mLeicosapentaenoic acid+0.5 μg/mL docosahexaenoic acid+0.5 μg/mLarachidonic acid+0.5 μg/mL myristic acid+1.85 μg/mL palmitic acid+2.5μg/mL stearic acid+0.25 μg/mL palmitoleic acid+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1.85 μg/mL oleic acid+1 μg/mL L-ascorbic acid 2-phosphatesesquimagnesium salt hydrate+0.1% Pluronic F-68+20 ng/mL bFGF+5 mg/mLtreated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+0.5μg/mL octadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+0.5 μg/mL myristicacid+1.85 μg/mL palmitic acid+2.5 μg/mL stearic acid+0.25 μg/mLpalmitoleic acid+25 μg/mL polyoxyethylenesorbitan monooleate+2 μg/mLD-alpha-tocopherol acetate+1.85 μg/mL oleic acid+1 μg/mL L-ascorbic acid2-phosphate sesquimagnesium salt hydrate+0.1% Pluronic F-68+20 ng/mLbFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+0.5μg/mL octadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+0.5 μg/mL myristicacid+1.85 μg/mL palmitic acid+ 2.5 μg/mL stearic acid+0.25 μg/mLpalmitoleic acid+1.85 μg/mL oleic acid+1 μg/mL L-ascorbic acid2-phosphate sesquimagnesium salt hydrate+0.1% Pluronic F-68+20 ng/mLbFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+0.5 μg/mL myristicacid+1.85 μg/mL palmitic acid+2.5 μg/mL stearic acid+0.25 μg/mLpalmitoleic acid+25 μg/mL polyoxyethylenesorbitan monooleate+2 μg/mLD-alpha-tocopherol acetate+1.85 μg/mL oleic acid+0.65 μg/mL linoleicacid+1 μg/mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+20ng/mL bFGF+5 mg/mL treated human serum albumin,

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+0.5 μg/mL alpha-linolenic acid+0.5 μg/mLgamma-linolenic acid+0.5 μg/mL dihomo-gamma-linolenic acid+0.5 μg/mLoctadecatetraenoic acid+0.5 μg/mL eicosapentaenoic acid+0.5 μg/mLdocosahexaenoic acid+0.5 μg/mL arachidonic acid+0.5 μg/mL myristicacid+1.85 μg/mL palmitic acid+2.5 μg/mL stearic acid+0.25 μg/mLpalmitoleic acid+25 μg/mL polyoxyethylenesorbitan monooleate+2 μg/mLD-alpha-tocopherol acetate+1.85 μg/mL oleic acid+1 μg/mL L-ascorbic acid2-phosphate sesquimagnesium salt hydrate+20 ng/mL bFGF+5 mg/mL treatedhuman serum albumin.

-   -   This variant, in which the treated human serum albumin was        treated by addition of 32 mM sodium octanoate, followed by        heating at 60 C for 4 h, followed by treatment with ion-exchange        resin (AG501-X8(D)) for 6 h at room temperature, followed by        treatment with dextran-coated activated charcoal (C6241,        Sigma-Aldrich Co. LLC.) overnight at room temperature, followed        by centrifugation, filtering, adjustment to a 10% solution with        nuclease-free water, followed by addition to the other        components of the medium, which was then conditioned for 24 h on        irradiated human neonatal fibroblast feeders, was used as the        transfection medium in Examples 2-7 and Examples 9-12, unless        otherwise noted, and cells were plated on fibronectin-coated        plates, unless otherwise noted. It is recognized that the        formulation of the transfection medium can be adjusted to meet        the needs of the specific cell types being cultured. It is        further recognized that treated human serum albumin can be        replaced with other treated albumin, for example, treated bovine        serum albumin, without negatively affecting the performance of        the medium. It is further recognized that other glutamine        sources can be used instead of or in addition to        L-alanyl-L-glutamine, for example, L-glutamine, that other        buffering systems can be used instead of or in addition to        HEPES, for example, phosphate, bicarbonate, etc., that selenium        can be provided in other forms instead of or in addition to        sodium selenite, for example, selenous acid, that other        antioxidants can be used instead of or in addition to L-ascorbic        acid 2-phosphate sesquimagnesium salt hydrate and/or        D-alpha-tocopherol acetate, for example, L-ascorbic acid, that        other surfactants can be used instead of or in addition to        polyoxyethylenesorbitan monooleate and/or Pluronic F-68, for        example, Pluronic F-127, that other basal media can be used        instead of or in addition to DMEM/F12, for example, MEM, DMEM,        etc., and that the components of the culture medium can be        varied with time, for example, by using a medium without TGF-β        from day 0 to day 5, and then using a medium containing 2 ng/mL        TGF-β after day 5, without negatively affecting the performance        of the medium. It is further recognized that other ingredients        can be added, for example, fatty acids, lysophosphatidic acid,        lysosphingomyelin, sphingosine-1-phosphate, other sphingolipids,        members of the TGF-β/NODAL family of proteins, IL-6, members of        the Wnt family of proteins, etc., at appropriate concentrations,        without negatively affecting the performance of the medium, and        that ingredients that are known to promote or inhibit the growth        of specific cell types and/or agonists and/or antagonists of        proteins or other molecules that are known to promote or inhibit        the growth of specific cell types can be added to the medium at        appropriate concentrations when it is used with those cell types        without negatively affecting the performance of the medium, for        example, sphingosine-1-phosphate and pluripotent stem cells. The        present invention relates equally to ingredients that are added        as purified compounds, to ingredients that are added as parts of        well-defined mixtures, to ingredients that are added as parts of        complex or undefined mixtures, for example, animal or plant        oils, and to ingredients that are added by biological processes,        for example, conditioning. The concentrations of the components        can be varied from the listed values within ranges that will be        obvious to persons skilled in the art without negatively        affecting the performance of the medium.

Example 9 Analysis of Transfection Efficiency and Viability of CellsCultured in Media Containing Ion-Exchange-Resin-Treated Human SerumAlbumin

Primary human neonatal fibroblasts were transfected according to Example2, using RNA synthesized according to Example 1, beginning on day 0.Pictures were taken on day 2 (FIG. 4). Cells in the well containinguntreated HSA (middle panel of FIG. 4) exhibited low viability comparedto either the well containing treated blood-derived HSA orion-exchange-resin-treated recombinant HSA.

Example 10 Reprogramming Human Fibroblasts UsingIon-Exchange-Resin-Treated Human Serum Albumin

Primary human neonatal fibroblasts were plated in 6-well plates onfeeders at a density of 10,000 cells/well in fibroblast medium (DMEM+10%fetal bovine serum). The cells were transfected according to Example 2,using RNA synthesized according to Example 1, beginning on day 0. Apassage with a split ratio of 1:20 was performed on day 4. Pictures weretaken on day 10. The well contained many large colonies of cellsexhibiting morphology consistent with reprogramming (FIG. 5). Nocolonies were observed in wells exposed to cell-culture media containinguntreated HSA.

Example 11 Reprogramming Human Fibroblasts without Using Feeders orImmunosuppressants

Primary human fibroblasts were plated in 6-well plates at a density of20,000 cells/well in fibroblast medium (DMEM+10% fetal bovine serum).After 6 hours, the medium was replaced with transfection mediumcontaining treated HSA and not containing immunosuppressants, and thecells were transfected as in Example 10, except that the dose of RNA wasreduced to 1 μg/well and the total number of transfections was reducedto 5. Pictures were taken on day 7 (FIG. 6). Small colonies of cellsexhibiting morphology consistent with reprogramming became visible asearly as day 5. On day 7, the medium was replaced with DMEM/F12+20%Knockout™ Serum Replacement (Life Technologies Corporation)+1×non-essential amino acids+2 mM L-glutamine, conditioned on irradiatedmouse embryonic fibroblasts for 24 hours, and then supplemented with 20ng/mL bFGF and 10 μM Y-27632. Large colonies with a reprogrammedmorphology became visible as early as day 8. Colonies were picked on day10, and plated in wells coated with basement membrane extract (Cultrex®Human BME Pathclear®, Trevigen Inc.) (FIG. 7). Cells grew rapidly, andwere passaged to establish lines. Established lines stained positive forthe pluripotent stem-cell markers Oct4 and SSEA4 (FIG. 8). The entireprotocol was repeated, and similar results were obtained (FIG. 9).

Example 12 Efficient, Rapid Derivation and Reprogramming of Cells fromHuman Skin Biopsy Tissue

A full-thickness dermal punch biopsy was performed on a healthy, 31year-old volunteer, according to an approved protocol. Briefly, an areaof skin on the left, upper arm was anesthetized by topical applicationof 2.5% lidocaine. The field was disinfected with 70% isopropanol, and afull-thickness dermal biopsy was performed using a 1.5 mm-diameter punch(FIG. 10A). The tissue was rinsed in phosphate-buffered saline (PBS),and was placed in a 1.5 mL tube containing 250 μL of TrypLE™ Select CTS™(Life Technologies Corporation), and incubated at 37 C for 30 min. Thetissue was then transferred to a 1.5 mL tube containing 250 μL ofDMEM/F12-CTS™ (Life Technologies Corporation)+5 mg/mL collagenase, andincubated at 37 C for 2 h (FIG. 10B). The epidermis was removed usingforceps, and the tissue was mechanically dissociated. Cells were rinsedtwice in DMEM/F12-CTS™ and were plated in fibronectin-coated wells of24-well and 96-well plates. Phlebotomy was also performed on the samevolunteer, and venous blood was collected in Vacutainer® SST™ tubes(Becton, Dickinson and Company). Serum was isolated according to themanufacturer's protocol. Isogenic plating medium was prepared by mixingDMEM/F12-CTS™+2 mM L-alanyl-L-glutamine (Sigma-Aldrich Co. LLC.)+20%human serum. Cells from the dermal tissue sample were plated either intransfection medium or in isogenic plating medium. After 2 days, thewells were rinsed, and the medium was replaced with transfection medium.Many cells with a fibroblast morphology attached and began to spread byday 2 (FIG. 10C). Cells were transfected according to Example 2, usingRNA synthesized according to Example 1, beginning on day 2, with allvolumes scaled to accommodate the smaller wells. By day 5, areas ofcells with morphologies consistent with reprogramming were observed(FIG. 11).

Example 13 Diabetes Disease Models for Screening Bioactive Molecules

Cells are reprogrammed according to Example 11 or Example 12, and arethen cultured in DMEM/F12+0.2% HSA+0.5×N2 supplement+0.5×B27supplement+100 ng/mL activin A+1 μM wortmannin for 4 days, followed by1:1 F12/IMDM+0.5% HSA+0.5% ITS supplement+0.5×B27 supplement+2 μMretinoic acid+20 ng/mL FGF7+50 ng/mL NOGGIN for 4 days, followed byDMEM+0.5% HSA+1% ITS supplement+1×N2 supplement+50 ng/mL EGF for 5 days,followed by DMEM/F12+1% ITS supplement+10 ng/mL bFGF+10 mMnicotinamide+50 ng/mL exendin-4+10 ng/mL BMP4 for 7-9 days to generateglucose-responsive insulin-producing cells. Alternatively, cells arereprogrammed according to Example 11 or Example 12, and are thencultured in 1:1 F12/IMDM+0.5% HSA+0.5% ITS supplement+0.5×B27supplement+2 μM retinoic acid+20 ng/mL FGF7+50 ng/mL NOGGIN for 4 days,followed by DMEM+0.5% HSA+1% ITS supplement+1×N2 supplement+50 ng/mL EGFfor 5 days, followed by DMEM/F12+1% ITS supplement+10 ng/mL bFGF+10 mMnicotinamide+50 ng/mL exendin-4+10 ng/mL BMP4 for 7-9 days to generateglucose-responsive insulin-producing cells, without generatingdefinitive endoderm cells. Alternatively, cells are reprogrammedaccording to Example 11 or Example 12, and are then cultured in 1:1F12/IMDM+0.5% HSA+0.5% ITS supplement+0.5×B27 supplement+2 μM retinoicacid+20 ng/mL FGF7+50 ng/mL NOGGIN for 4 days, followed by DMEM/F12+1%ITS supplement+10 ng/mL bFGF+10 mM nicotinamide+50 ng/mL exendin-4+10ng/mL BMP4 for 7-9 days to generate glucose-responsive insulin-producingcells, without generating definitive endoderm cells, and withoutexpanding progenitor cells. While endodermal cells or insulin-producingcells can be isolated from other cells present in the culture, thismethod generates a sufficiently high percentage of glucose-responsiveinsulin producing cells that such isolation is not generally required.The resulting cells can then be used in vitro or in vivo for screeningbioactive molecules for the study of diabetes or for the development oftherapeutics for diabetes.

Example 14 Personalized Cell-Replacement Therapy for Type 1 DiabetesComprising Reprogrammed Cells

Patient skin cells are reprogrammed to glucose-responsiveinsulin-producing cells according to Example 12 and Example 13. Cellsare then enzymatically released from the culture vessel, and betweenabout 1×10⁶ and about 1×10⁷ cells are injected into the intraperitonealspace or into the portal vein. In the case of intraperitoneal injection,cells are pre-mixed with an extracellular matrix protein to preventexcessive migration. Cells engraft and begin producing insulin.Insulin/C-peptide levels are monitored, and additional injections areperformed as necessary.

Example 15 Cardiac Disease Models for Screening Bioactive Molecules

Cells were reprogrammed according to Example 11, and were then culturedin DMEM/F12+0.2% HSA+0.5×N2 supplement+0.5×B27 supplement+100 ng/mLactivin A+1 μM wortmannin for 4 days, followed by 1:1 F12/IMDM+0.5%HSA+0.5% ITS supplement+0.5×B27 supplement+2 μM retinoic acid+20 ng/mLFGF7+50 ng/mL NOGGIN for 4 days, followed by DMEM/F12+1% ITSsupplement+10 ng/mL bFGF+10 mM nicotinamide+50 ng/mL exendin-4+10 ng/mLBMP4 for 7-9 days to generate cardiac cells (FIG. 12). While cardiaccells can be isolated from other cells present in the culture, thismethod generates a sufficiently high percentage of cardiac cells thatsuch isolation is not generally required. The resulting cells can beused in vitro or in vivo for screening bioactive molecules for the studyof heart disease or for the development of therapeutics for heartdisease. The resulting cells can also be used for cardiotoxicityscreening.

Example 16 Personalized Cell-Replacement Therapy for IschemicCardiomyopathy Comprising Reprogrammed Cells

Patient skin cells are reprogrammed to cardiac cells according toExample 15. Cells are then enzymatically released from the culturevessel, and between about 1×10⁶ and about 1×10⁷ cells are injected intothe pericardium or between about 1×10³ and about 1×10⁵ cells areinjected into one or more coronary arteries. Cells engraft, andadditional injections are performed as necessary.

Example 17 Personalized Cell-Replacement Therapy for Blood DiseaseComprising Reprogrammed Cells

Cells are reprogrammed according to Example 11 or Example 12, and arethen cultured in IMDM+0.5% HSA+1×ITS supplement+450 μMmonothioglycerol+2 mM L-glutamine+1× non-essential amino acids+50 ng/mLBMP4+50 ng/mL VEGF+50 ng/mL bFGF for 6 days, followed by IMDM+0.5%HSA+1×ITS supplement+0.1 mM 2-mercaptoethanol+5 U/mL heparin+10 ng/mLTPO+25 ng/mL SCF+25 ng/mL FLT3L+10 ng/mL IL-3+10 ng/mL IL-6 for 8 daysto generate hematopoietic cells. Alternatively, cells are reprogrammedaccording to Example 11 or Example 12, and are then re-plated oncollagen IV and cultured in IMDM+0.5% HSA+1×ITS supplement+450 μMmonothioglycerol+2 mM L-glutamine+1× non-essential amino acids+50 ng/mLBMP4+50 ng/mL VEGF+50 ng/mL bFGF for 6 days, followed by IMDM+0.5%HSA+1×ITS supplement+0.1 mM 2-mercaptoethanol+5 U/mL heparin+10 ng/mLTPO+25 ng/mL SCF+25 ng/mL FLT3L+10 ng/mL IL-3+10 ng/mL IL-6 for 8 daysto generate hematopoietic cells. Alternatively, cells are reprogrammedaccording to Example 11 or Example 12, and are then cultured in 1:1F12/IMDM+0.5% HSA+1×ITS supplement+4.5 μg/mL cholesterol+10 μg/mL codliver oil fatty acids+25 μg/mL polyoxyethylenesorbitan monooleate+2μg/mL D-α-tocopherol acetate+450 μM monothioglycerol+2 mM L-glutamine+25ng/mL BMP4+25 ng/mL VEGF+25 ng/mL bFGF+20 ng/mL SCF for 10 days togenerate hematopoietic cells.

Example 18 Personalized Cell-Replacement Therapy for Blood DiseaseComprising Reprogrammed Cells

Patient skin cells are reprogrammed to hematopoietic cells according toExample 17. Cells are then released from the culture vessel, and betweenabout 1×10⁶ and about 1×10⁷ cells/kg patient body weight are infusedinto a main vein over a period of several hours.

Example 19 Retinal Disease Models for Screening Bioactive Molecules

Cells are reprogrammed according to Example 11 or Example 12, and arethen cultured in DMEM/F12+0.2% HSA+0.5×N2 supplement+0.5×B27 supplement7 days to generate retinal cells. The resulting cells can be used invitro or in vivo for screening bioactive molecules for the study ofretinal disease or for the development of therapeutics for retinaldisease.

Example 20 Personalized Cell-Replacement Therapy for MacularDegeneration Comprising Reprogrammed Cells

Patient skin cells are reprogrammed to retinal cells according toExample 15. Cells are then enzymatically released from the culturevessel, and between about 1×10⁴ and about 1×10⁵ cells are injected intoor below the retina. Cells engraft, and additional injections areperformed as necessary.

REFERENCES

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What is claimed is:
 1. A method for reprogramming a cell to a lessdifferentiated state, comprising: a. culturing a differentiated cellwith a reprogramming medium containing albumin, wherein the albumin istreated with an ion-exchange resin or charcoal; b. transfecting the cellwith one or more synthetic RNA molecules, wherein the one or moresynthetic RNA molecules includes at least one RNA molecule encoding oneor more reprogramming factors and wherein the transfecting results inthe cell expressing the one or more reprogramming factors; and c.repeating step (b) at least twice during 5 consecutive days to result inthe cell being reprogrammed to a less differentiated state.
 2. Themethod of claim 1, wherein the albumin was treated with sodiumoctanoate.
 3. The method of claim 2, wherein the albumin was brought toa temperature of at least 40° C.
 4. The method of claim 1, wherein theone or more synthetic RNA molecules includes at least one RNA moleculeencoding at least one member of the group: Oct4 protein, Sox2 protein,Klf4 protein, and c-Myc protein.
 5. The method of claim 1, wherein theone or more synthetic RNA molecules includes at least one RNA moleculeencoding Oct4 protein, at least one RNA molecule encoding Sox2 protein,at least one RNA molecule encoding Klf4 protein, and at least one RNAmolecule encoding c-Myc protein.
 6. The method of claim 1, furthercomprising contacting the cell with at least one member of the group:poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin,collagen, and laminin.
 7. The method of claim 1, wherein the one or moresynthetic RNA molecules contains at least one member of the group: apseudouridine residue and a 5-methylcytidine residue.
 8. The method ofclaim 1, wherein the cell is a skin cell.
 9. A method for reprogramminga cell to a less differentiated state, comprising: a. culturing adifferentiated cell with a reprogramming medium containing albumin,wherein the albumin is treated with sodium octanoate; brought to atemperature of at least 40° C.; and treated with an ion-exchange resinor charcoal; b. transfecting the cell with one or more synthetic RNAmolecules, wherein the one or more synthetic RNA molecules includes atleast one RNA molecule encoding one or more reprogramming transcriptionfactors and wherein the transfecting results in the cell expressing theone or more synthetic RNA molecules; and c. repeating step (b) at leasttwice during 5 consecutive days to result in the cell being reprogrammedto a less differentiated state.
 10. The method of claim 9, wherein theone or more synthetic RNA molecules includes at least one RNA moleculeencoding at least one member of the group: Oct4 protein, Sox2 protein,Klf4 protein, and c-Myc protein.
 11. The method of claim 9, wherein oneor more synthetic RNA molecules includes at least one RNA moleculeencoding Oct4 protein, at least one RNA molecule encoding Sox2 protein,at least one RNA molecule encoding Klf4 protein, and at least one RNAmolecule encoding c-Myc protein.
 12. The method of claim 9, furthercomprising contacting the cell with at least one member of the group:poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin,collagen, and laminin.
 13. The method of claim 9, wherein the one ormore synthetic RNA molecules contains at least one member of the group:a pseudouridine residue and a 5-methylcytidine residue.
 14. The methodof claim 9, wherein the cell is a skin cell.